PCSK9 vaccine

ABSTRACT

The present invention relates to the provision of immunogens comprising an antigenic PCSK9 peptide linked to an immunogenic carrier for the prevention, treatment or alleviation of PCSK9-mediated disorders. The invention further relates to methods for production of these medicaments, immunogenic compositions and pharmaceutical compositing thereof and their use in medicine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/872,645filed on Aug. 31, 2010, now U.S. Pat. No. 8,889,144, which claimsbenefit of U.S. Provisional Application No. 61/239,541 filed on Sep. 3,2009. Both application Ser. No. 12/872,645 and U.S. ProvisionalApplication No. 61/239,541 are incorporated herein by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence entitled“PC33896B_UpdatedSequenceListing_ST25.txt” created on Jun. 6, 2016 andhaving a size of 121 KB. The sequence listing contained in this .txtfile is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the provision of novel immunogenscomprising an antigenic PCSK9 peptide preferably linked to animmunogenic carrier for the prevention, treatment or alleviation ofPCSK9-related disorders. The invention further relates to methods forproduction of these medicaments, immunogenic compositions andpharmaceutical composition thereof and their use in medicine.

BACKGROUND

Proprotein convertase subtilisin-kexin type 9 (hereinafter called“PCSK9”), also known as neural apoptosis-regulated convertase 1(“NARC-I”), is a proteinase K-like subtilase identified as the 9thmember of the mammalian PCSK family; see Seidah et al, 2003 PNAS100:928-933. The gene for PCSK9 localizes to human chromosome1p33-p34.3. PCSK9 is expressed in cells capable of proliferation anddifferentiation including, for example, hepatocytes, kidney mesenchymalcells, intestinal ileum, and colon epithelia as well as embryonic braintelencephalon neurons.

Original synthesis of PCSK9 is in the form of an inactive enzymeprecursor, or zymogen, of ˜72-kDa which undergoes autocatalytic,intramolecular processing in the endoplasmic reticulum (“ER”) toactivate its functionality. The gene sequence for human PCSK9, which is˜22-kb long with 12 exons encoding a 692 amino acid protein, can befound, for example, at Deposit No. NP_777596.2. Human, mouse and ratPCSK9 nucleic acid sequences have been deposited; see, e.g., GenBankAccession Nos.: AX127530 (also AX207686), AX207688, and AX207690,respectively.

Human PCSK9 is a secreted protein expressed primarily in the kidneys,liver and intestines. It has three domains: an inhibitory pro-domain(amino acids 1-152; including a signal sequence at amino acids 1-30), acatalytic domain (amino acids 153-448), and a C-terminal domain 210residues in length (amino acids 449-692), which is rich in cysteineresidues. PCSK9 is synthesized as a zymogen that undergoes autocatalyticcleavage between the pro-domain and catalytic domain in the endoplasmicreticulum. The pro-domain remains bound to the mature protein aftercleavage, and the complex is secreted. The cysteine-rich domain may playa role analogous to the P-(processing) domains of otherFurin/Kexin/Subtilisin-like serine proteases, which appear to beessential for folding and regulation of the activated protease.Mutations in PCSK9 are associated with abnormal levels of low densitylipoprotein cholesterol (LDL-c) in the blood plasma (Horton et al., 2006Trends. Biochem. Sci. 32(2):71-77).

PCSK9 has been ascribed a role in the differentiation of hepatic andneuronal cells (Seidah et al, supra), is highly expressed in embryonicliver, and has been strongly implicated in cholesterol homeostasis.

The identification of compounds and/or agents effective in the treatmentof cardiovascular affliction is highly desirable. Reductions in LDLcholesterol levels have already demonstrated in clinical trials to bedirectly related to the rate of coronary events; Law et al, 2003 BMJ326: 1423-1427. More, recently moderate lifelong reduction in plasma LDLcholesterol levels has been shown to be substantially correlated with asubstantial reduction in the incidence of coronary events; Cohen et al,supra. This was found to be the case even in populations with a highprevalence of non-lipid-related cardiovascular risk factors; supra.

Accordingly, it is of great importance to identify therapeutic agentpermitting the control of LDL cholesterol levels.

Accordingly, it would be of great importance to produce a medicamentthat inhibits or antagonizes the activity of PCSK9 and the correspondingrole PCSK9 plays in various therapeutic conditions.

Expression or upregulation of PCSK9 is associated with increased plasmalevels of LDL cholesterol, and inhibition or the lack of expression ofPCSK9 is associated with low LDL cholesterol plasma levels.Significantly, lower levels of LDL cholesterol associated with sequencevariations in PCSK9 have conferred protection against coronary heartdisease; Cohen, 2006 N. Engl. J. Med. 354: 1264-1272.

SUMMARY OF THE INVENTION

The present invention relates to an immunogen comprising an antigenicPCSK9 peptide and optionally an immunogenic carrier.

The invention also relates to methods for producing such antigenic PCSK9peptide optionally linked to an immunogenic carrier.

The invention also relates to immunogenic compositions comprising suchantigenic PCSK9 peptide optionally linked to an immunogenic carrier,optionally comprising one or several adjuvants, preferably one or twoadjuvants.

Another aspect of the invention relates to pharmaceutical compositionscomprising an antigenic PCSK9 peptide according to the invention, or animmunogenic composition thereof, as well as to medical uses of suchcompositions.

In particular, the invention relates to an antigenic PCSK9 peptide ofthe invention, or an immunogenic or pharmaceutical composition thereof,for use as a medicament, preferably in treatment, alleviation orprophylaxis of PCSK9-related disorders.

In particular, the invention relates to an antigenic PCSK9 peptide ofthe invention, or an immunogenic or pharmaceutical composition thereof,for use as a medicament preferably in treatment, alleviation orprophylaxis of diseases associated with an elevated level ofcholesterol.

The antigenic PCSK9 peptides of the invention are particularly suitablefor treating human patients having, or at risk for, elevatedLDL-cholesterol or a condition associated with elevated LDL-cholesterol,e.g., a lipid disorder (e.g., hyperlipidemia, type I, type II, type III,type IV, or type V hyperlipidemia, secondary hypertriglyceridemia,hypercholesterolemia, familial hypercholesterolemia, xanthomatosis,cholesterol acetyltransferase deficiency). Antigenic PCSK9 peptide ofthe invention are also suitable for treating human patients havingarteriosclerotic conditions (e.g., atherosclerosis), coronary arterydisease, cardiovascular disease, and patients at risk for thesedisorders, e.g., due to the presence of one or more risk factors (e.g.,hypertension, cigarette smoking, diabetes, obesity, orhyperhomocysteinemia).

In yet another aspect, the present invention provides the use of anantigenic PCSK9 peptide of the invention or of an immunogeniccomposition or a pharmaceutical composition thereof, in the manufactureof a medicament for the treatment of Alzheimer's disease.

In one embodiment, the antigenic PCSK9 peptide or an immunogeniccomposition or a pharmaceutical composition thereof is administeredtogether with another agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The PDB structure of human PCSK9 bound to the EGF-A domain ofthe LDL-R (3BPS) showing 5 peptide sequences in PCSK9 (Peptides 1-5)chosen as being involved in the interaction between these two proteins.The amino acid sequence of each of these peptides is set forth below:

Peptide 1: ASSDCSTCFV (SEQ ID NO:19);

Peptide 2: GTRFHRQASKC (SEQ ID NO:62);

Peptide 3: SDHREIEGRV (SEQ ID NO:121);

Peptide 4: SGRDAGVAKGA (SEQ ID NO:153);

Peptide 5: SIPWNLERITP (SEQ ID NO:184).

FIG. 2: Mice were immunized with peptides VR_9.1 to VR_9.9 conjugated toVLPs, using Alum plus CpG as adjuvant and antibody responses tofull-length recombinant human PCSK9 were measured by titrating sera inan ELISA assay. Results are shown as reciprocal titres for each of 6mice per group, with the reciprocal titre measured as the dilution ofserum giving an optical density reading of 0.5.

FIG. 3: Antibody responses to full-length recombinant mouse PCSK9protein as described in FIG. 2.

FIG. 4: Plasma cholesterol levels measured in the sera of vaccinatedmice (same samples as used for antibody assays in FIGS. 2 and 3).

FIG. 5: Plasma samples used in FIGS. 2 to 4 were tested at differentdilutions for their ability to inhibit the interaction betweenrecombinant PCSK9 and the extracellular domain of the LDL receptor asmeasured by FRET assay.

FIG. 6: Dilutions of plasma samples from peptides VR_9.5 and VR_9.6vaccinations in FRET assay, showing dose-responsive inhibition ofinteraction between PCSK9 and the LDL receptor.

FIG. 7: Complex of PCSK9 (ribbons) and EGF-A (space fill) from PDB:3BPS.Potential regions of PCSK9 that may interact with domains of LDLR otherthan EGF-A are indicated by the ellipse.

FIG. 8: Complex of PCSK9 (ribbons) and EGF-A (space fill), with theamino acids corresponding to peptides VR_13/14 (A) and VR_15/16 (B) andVR_9.5 (C) is displayed.

FIG. 9: Plasma antibody responses of mice vaccinated with peptidesVR_9.5 (upper left panel), VR_9.10 (upper middle panel), VR_9.11 (upperright panel), VR_9.12 (lower left panel), and VR_9.13 (lower rightpanel). Antibodies to mouse PCSK9 were measured by ELISA assay of serialplasma dilutions using full-length mouse PCSK9 protein. Individualtitration curves are shown for 8 mice per group, with ELISA responses ofplasma from mice immunized with unconjugated VLP shown as a control.

FIG. 10: Plasma antibody responses of mice vaccinated with peptidesVR_9.14 (upper left panel), VR_9.15 (upper right panel), VR_9.16 (lowerleft panel), and Control VLP (lower right panel). Antibodies to mousePCSK9 were measured by ELISA assay of serial plasma dilutions usingfull-length mouse PCSK9 protein. Individual titration curves are shownfor 8 mice per group, with ELISA responses of plasma from mice immunizedwith unconjugated VLP shown as a control.

FIG. 11: Serum antibody responses to full-length human PCSK9 proteininduced in BALB/c mice (left panel) and C57BL/6 mice (right panel)vaccinated with either peptide VR_9.5 or VR_9.10 conjugated to VLPs(using Alum+/−CpG as adjuvant) or CRM197 (using TiterMax as adjuvant).Antibodies to human PCSK9 were measured by titrating sera in an ELISAassay. Results are shown as log reciprocal titres determined at anoptical density of 1.0 for each of 8 mice per group.

FIG. 12: Serum antibody responses to full-length mouse PCSK9 proteininduced in BALB/c mice (left panel) and C57BL/6 mice (right panel)vaccinated as described for FIG. 11. Results are shown as log reciprocaltitres determined at an optical density of 0.5 for each of 8 mice pergroup.

FIG. 13: Total cholesterol levels measured in the serum samples fromBALB/c vaccinated mice (same samples used for antibody assays in FIGS.11 and 12).

FIG. 14: Total cholesterol levels measured in the serum samples fromC57BL/6 vaccinated mice (same samples used for antibody assays in FIGS.11 and 12).

FIG. 15: Antibody responses to full-length human PCSK9 induced in BALB/cmice immunized with peptides VR_9.5 or VR_9.17 to VR_9.35 conjugated toVLPs using Alum plus CpG as adjuvant. Antibodies to human PCSK9 weremeasured by titrating sera in an ELISA assay. Results are shown as logreciprocal titres determined at an optical density of 1.0 for each of 8mice per group.

FIG. 16: Antibody responses to full-length mouse PCSK9 induced in BALB/cmice immunized as described for FIG. 15. Antibodies to mouse PCSK9 weremeasured by titrating sera in an ELISA assay. Results are shown as logreciprocal titres determined at an optical density of 0.5 for each of 8mice per group.

FIG. 17: Total cholesterol levels measured in the serum samples fromBALB/c vaccinated mice (same samples used for antibody assays in FIGS.15 and 16).

FIG. 18: Complex of PCSK9 (ribbons) and EGF-A (space fill) with regionsof PCSK9 containing amino acids sequences linked to gain- orloss-of-function mutations and/or protein surface exposed epitopesindicated by ellipses.

FIG. 19: Diagram illustrating linkage of an antigenic peptide to acarrier.

DETAILED DESCRIPTION OF THE INVENTION Antigenic PCSK9 Peptide of theInvention

The present invention relates to an immunogen comprising an antigenicPCSK9 peptide optionally linked to an immunogenic carrier.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9comprising between 4 to 20 amino acids and, when administered to asubject, is able to lower the LDL-cholesterol level in blood of saidsubject. Preferably, said subject is a mammal, preferably a human.Preferably, said antigenic PCSK9 peptide is able to lower theLDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9which participates in the interaction of PCSK9 with the LDL receptor.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9which participates in the interaction of PCSK9 with the LDL receptor,comprising between 4 and 20 amino acids and, when administered to asubject is able to lower the LDL-cholesterol level in blood of saidsubject. Preferably, said subject is a mammal, preferably a human.Preferably, said antigenic PCSK9 peptide is able to lower theLDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503,504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517,518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559,560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573,574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587 and588.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 1 to 312, 330 to 398, 421, 423, 424, 426and 428 to 588.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 and 398.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9which may participate in the interaction with the domain EGF-A of theLDL receptor. Examples of such portions are represented on FIG. 1.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9which may participate in the interaction with the domain EGF-A of theLDL receptor, comprising between 4 and 20 amino acids and, whenadministered to a subject, is able to lower the LDL-cholesterol level inblood of said subject. Preferably, said subject is a mammal, preferablya human. Preferably, said antigenic PCSK9 peptide is able to lower theLDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 1.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 15, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 46.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100 and 101.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 14, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 102.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145 and 146.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 147.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 and 181.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 182.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225 and 226.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 330.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 330, 331, 332, 333, 334, 335, 336, 337,338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358 and 359.

In a preferred embodiment, the antigenic PCSK9 peptide is selected fromthe group consisting of SEQ ID Nos 19, 56, 63, 109, 153, 165, 184, 186,187, 188, 332 and 424.

In a preferred embodiment, the antigenic PCSK9 peptide is selected fromthe group consisting of SEQ ID Nos 19, 56, 63, 109, 153 and 184.

In a preferred embodiment, the antigenic PCSK9 peptide is selected fromthe group consisting of SEQ ID Nos 56, 184, 186, 187, 188 and 332.

In a most preferred embodiment, the antigenic PCSK9 peptide is a peptideof sequence SEQ ID No 56.

In a more preferred embodiment, the antigenic PCSK9 peptide is a peptideof sequence SEQ ID No 184 or 187.

In a most preferred embodiment, the antigenic PCSK9 peptide is a peptideof sequence SEQ ID No 184.

In a most preferred embodiment, the antigenic PCSK9 peptide is a peptideof sequence SEQ ID No 332.

In one embodiment, the antigenic PCSK9 peptide is selected in a regionof PCSK9 which may participate in the interaction with a region of theLDL receptor other than the EGF-A domain. Examples of such portions arerepresented on FIGS. 7 and 8.

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9which may participate in the interaction with a region of the LDLreceptor other than the EGF-A domain, comprising between 4 and 20 aminoacids and, when administered to a subject, is able to lower theLDL-cholesterol level in blood of said subject. Preferably, said subjectis a mammal, preferably a human. Preferably, said antigenic PCSK9peptide is able to lower the LDL-cholesterol level by at least 2%, 5%,10%, 20%, 30% or 50%.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 12, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 227.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 and 262.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 263.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306 and 307.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 360.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397 and 398.

In an embodiment, the antigenic PCSK9 peptide is selected in a region ofPCSK9 pro-domain (SEQ ID No 329).

In one embodiment, the antigenic PCSK9 peptide is a portion of PCSK9pro-domain, comprising between 4 and 20 amino acids and, whenadministered to a subject, is able to lower the LDL-cholesterol level inblood of said subject. Preferably, said subject is a mammal, preferablya human. Preferably, said antigenic PCSK9 peptide is able to lower theLDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 308, 309, 310, 311 and 312.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 12, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 309.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 309, 429, 430, 431, 432, 433, 434, 435,436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 and 463.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 12, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 508.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 508, 509, 510, 511, 512, 513, 514, 515,516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529,530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542 and 543.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 310.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 310, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500, 501, 502, 503, 504, 505, 506 and 507.

In one embodiment, the antigenic PCSK9 peptide is a peptide comprising 5to 13, preferably 6 to 8, consecutive amino acids of the PCSK9 fragmentof SEQ ID No 544.

In one embodiment, the antigenic PCSK9 peptide is selected from thegroup consisting of SEQ ID Nos 544, 545, 546, 547, 548, 549, 550, 551,552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565,566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,580, 581, 582, 583, 584, 585, 586, 587, and 588.

In a preferred embodiment, the antigenic PCSK9 peptide is selected fromthe group consisting of SEQ ID Nos 312, 421, 422, 423, 426, 427, 428,445, 482, 525, and 563.

In a more preferred embodiment, the antigenic PCSK9 peptide is selectedfrom the group consisting of SEQ ID Nos 445, 482, 525, and 563.

In a most preferred embodiment, the antigenic PCSK9 peptide is a peptideof sequence SEQ ID No 445.

Such antigenic PCSK9 peptides may be used alone or in combination,preferably when conjugated to an immunogenic carrier, to induce autoanti-PCSK9 antibodies in a subject in order to treat, prevent orameliorate PCSK9-related disorders.

It will be apparent to the man skilled in the art which techniques maybe used to confirm whether a specific construct falls within the scopeof the present invention. Such techniques include, but are notrestricted to, the techniques described in the Example section of thepresent application, and also to the following.

The ability of the antigenic PCSK9 peptide of the invention to induceauto anti-PCSK9 antibodies may be measured in mice, using the testdisclosed in example 3 of the present application. The ability ofauto-antibodies induced by the antigenic PCSK9 peptide of the inventionto decrease the level of circulating plasma cholesterol may be measuredin mice, using the test disclosed in example 3. The ability ofauto-antibodies induced by the antigenic PCSK9 peptide of the inventionto inhibit the interaction between PCSK9 and LDL receptors may bemeasured directly using the test disclosed in example 3 (FRET assay) orindirectly by measuring the upregulation of cell surface LDL receptorswhich is a consequence of blocking PCSK9-mediated down-regulation (aswell described in the relevant literature, either using cell lines invitro or by measuring LDL receptor levels in liver biopsies of antibodyexpressing animals (e.g. by Western blotting)).

The term “antigenic PCSK9 peptide biological activity”, when usedherein, refers to the ability of the antigenic PCSK9 peptides of theinvention to induce auto anti-PCSK9 antibodies in a patient.

Preferably said antigenic PCSK9 peptide, when administered to a subject,is able to lower the LDL-cholesterol level in blood of said subject.Preferably, said subject is a mammal, preferably a human. Preferably,said antigenic PCSK9 peptide is able to lower the LDL-cholesterol levelby at least 2%, 5%, 10%, 20%, 30% or 50%.

In an embodiment the antigenic PCSK9 peptides of the present inventionare of a size such that they mimic a region selected from the wholePCSK9 domain in which the native epitope is found. In a particularembodiment, the antigenic PCSK9 peptides of the invention are less than100 amino acids in length, preferably shorter than 75 amino acids, morepreferably less than 50 amino acids, even more preferably less than 40amino acids. The antigenic PCSK9 peptides of the invention are typically4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length, preferably from4 to 20 amino acids, for example 6 to 12, 6 to 8 or 9 to 12 amino acids.

Specific examples of antigenic PCSK9 peptides of the invention areprovided in the sequence listing and include peptides ranging from 5 to17 amino acids in length.

The antigenic peptides of the invention include an amino acid sequencederived from a portion of a mammalian PCSK9, preferably a human PCSK9(SEQ ID No 399) or mouse PCSK9 (SEQ ID Nos 400), more preferably humanPCSK9, such derived portion of PCSK9 either corresponding to the aminoacid sequence of naturally occurring PCSK9 or corresponding to variantPCSK9, i.e. the amino acid sequence of naturally occurring PCSK9 inwhich a small number of amino acids have been substituted, added ordeleted but which retains essentially the same immunological properties.In addition, such derived PCSK9 portion can be further modified by aminoacids, especially at the N- and C-terminal ends to allow the antigenicPCSK9 peptide to be conformationally constrained and/or to allowcoupling of the antigenic PCSK9 peptide to an immunogenic carrier afterappropriate chemistry has been carried out.

The antigenic PCSK9 peptides disclosed herein encompass functionallyactive variant peptides derived from the amino acid sequence of PCSK9 inwhich amino acids have been deleted, inserted or substituted withoutessentially detracting from the immunological properties thereof, i.e.such functionally active variant peptides retain a substantial antigenicPCSK9 peptide biological activity. Typically, such functionally variantpeptides have an amino acid sequence homologous, preferably highlyhomologous, to an amino acid sequence selected from the group consistingof SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588.

In one embodiment, such functionally active variant peptides exhibit atleast 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to an amino acidsequence selected from the group consisting of SEQ ID Nos: 1 to 312, 330to 398 and 420 to 588.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). An alternative algorithm when comparing a sequenceof the invention to a database containing a large number of sequencesfrom different organisms is the computer program BLAST, especiallyblastp or tblastn, using default parameters. See, e.g., Altschul et al.,J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997).

Functionally active variants comprise naturally occurring functionallyactive variants such as allelic variants and species variants andnon-naturally occurring functionally active variants that can beproduced by, for example, mutagenesis techniques or by direct synthesis.

A functionally active variant differs by about, for example, 1, 2, 3, 4or 5 amino acid residues from any of the peptide shown at SEQ ID Nos: 1to 312, 330 to 398 and 420 to 588, and yet retain an antigenic PCSK9biological activity. Where this comparison requires alignment thesequences are aligned for maximum homology. The site of variation canoccur anywhere in the peptide, as long as the biological activity issubstantially similar to a peptide shown in SEQ ID Nos: 1 to 312, 330 to398 and 420 to 588.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al., Science, 247: 1306-1310(1990), which teaches that there are two main strategies for studyingthe tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, the amino acid positions which havebeen conserved between species can be identified. These conserved aminoacids are likely important for protein function. In contrast, the aminoacid positions in which substitutions have been tolerated by naturalselection indicate positions which are not critical for proteinfunction. Thus, positions tolerating amino acid substitution can bemodified while still maintaining specific immunogenic activity of themodified peptide.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site-directed mutagenesis oralanine-scanning mutagenesis can be used (Cunningham et al., Science,244: 1081-1085 (1989)). The resulting variant peptides can then betested for specific antigenic PCSK9 biological activity.

According to Bowie et al., these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,the most buried or interior (within the tertiary structure of theprotein) amino acid residues require nonpolar side chains, whereas fewfeatures of surface or exterior side chains are generally conserved.

Methods of introducing a mutation into amino acids of a protein is wellknown to those skilled in the art. See, e. g., Ausubel (ed.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y.(1989)).

Mutations can also be introduced using commercially available kits suchas “QuikChange™ Site-Directed Mutagenesis Kit” (Stratagene) or directlyby peptide synthesis. The generation of a functionally active variant toan antigenic PCSK9 peptide by replacing an amino acid which does notsignificantly influence the function of said antigenic PCSK9 peptide canbe accomplished by one skilled in the art.

A type of amino acid substitution that may be made in one of thepeptides according to the invention is a conservative amino acidsubstitution. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent sequence identityor degree of similarity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. See e.g.Pearson, Methods Mol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992). A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

A functionally active variant peptide can also be isolated using ahybridization technique. Briefly, DNA having a high homology to thewhole or part of a nucleic acid sequence encoding the peptide ofinterest, e.g. SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588, is usedto prepare a functionally active peptide. Therefore, an antigenic PCSK9peptide of the invention also includes peptides which are functionallyequivalent to one or more of the peptide of SEQ ID Nos: 1 to 312, 330 to398, and 420 to 588 and which are encoded by a nucleic acid moleculewhich hybridizes with a nucleic acid encoding any one of SEQ ID Nos: 1to 312, 330 to 398 and 420 to 588, or a complement thereof. One of skillin the art can easily determine nucleic acid sequences that encodepeptides of the invention using readily available codon tables. As such,these nucleic acid sequences are not presented herein.

The stringency of hybridization for a nucleic acid encoding a peptide,polypeptide or protein that is a functionally active variant is, forexample, 10% formamide, 5×SSPE, 1× Denhart's solution, and 1× salmonsperm DNA (low stringency conditions). More preferable conditions are,25% formamide, 5×SSPE, 1× Denhart's solution, and 1× salmon sperm DNA(moderate stringency conditions), and even more preferable conditionsare, 50% formamide, 5×SSPE, 1× Denhart's solution, and 1× salmon spermDNA (high stringency conditions). However, several factors influence thestringency of hybridization other than the above-described formamideconcentration, and one skilled in the art can suitably select thesefactors to accomplish a similar stringency.

Nucleic acid molecules encoding a functionally active variant can alsobe isolated by a gene amplification method such as PCR using a portionof a nucleic acid molecule DNA encoding a peptide, polypeptide orprotein of interest, e.g. any one of the peptides shown SEQ ID Nos: 1 to312, 330 to 398 and 420 to 588, as the probe.

In one embodiment of the invention, a peptide of the invention isderived from a natural source and isolated from a mammal, such as ahuman, a primate, a cat, a dog, a horse, a mouse, or a rat, preferablyfrom a human source. A peptide of the invention can thus be isolatedfrom cells or tissue sources using standard protein purificationtechniques. Alternatively, peptides of the invention can be synthesizedchemically or produced using recombinant DNA techniques.

For example, a peptide of the invention can be synthesized by solidphase procedures well known in the art. Suitable syntheses may beperformed by utilising “T-boc” or “F-moc” procedures. Cyclic peptidescan be synthesised by the solid phase procedure employing the well-known“F-moc” procedure and polyamide resin in the fully automated apparatus.Alternatively, those skilled in the art will know the necessarylaboratory procedures to perform the process manually. Techniques andprocedures for solid phase synthesis are described in ‘Solid PhasePeptide Synthesis: A Practical Approach’ by E. Atherton and R. C.Sheppard, published by IRL at Oxford University Press (1989) and‘Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed.M. W. Pennington and B. M. Dunn), chapter 7, pp 91-171 by D. Andreau etal.

Alternatively, a polynucleotide encoding a peptide of the invention canbe introduced into an expression vector that can be expressed in asuitable expression system using techniques well known in the art,followed by isolation or purification of the expressed peptide,polypeptide, or protein of interest. A variety of bacterial, yeast,plant, mammalian, and insect expression systems are available in the artand any such expression system can be used. Optionally, a polynucleotideencoding a peptide of the invention can be translated in a cell-freetranslation system.

Antigenic PCSK9 peptides of the invention can also comprise those thatarise as a result of the existence of multiple genes, alternativetranscription events, alternative RNA splicing events, and alternativetranslational and postranslational events. A peptide can be expressed insystems, e.g. cultured cells, which result in substantially the samepostranslational modifications present as when the peptide is expressedin a native cell, or in systems that result in the alteration oromission of postranslational modifications, e.g. glycosylation orcleavage, present when expressed in a native cell.

An antigenic PCSK9 peptide of the invention can be produced as a fusionprotein that contains other non-PCSK9 or non-PCSK9-derived amino acidsequences, such as amino acid linkers or signal sequences or immunogeniccarriers as defined herein, as well as ligands useful in proteinpurification, such as glutathione-S-transferase, histidine tag, andstaphylococcal protein A. More than one antigenic PCSK9 peptide of theinvention can be present in a fusion protein. The heterologouspolypeptide can be fused, for example, to the N-terminus or C-terminusof the peptide of the invention. A peptide of the invention can also beproduced as fusion proteins comprising homologous amino acid sequences,i. e., other PCSK9 or PCSK9-derived sequences.

The antigenic PCSK9 peptides of the invention might be linear orconformationally constrained. As used herein in reference to a peptide,the term “conformationally constrained” means a peptide, in which thethree-dimensional structure is maintained substantially in one spatialarrangement over time. Conformationally constrained molecules can haveimproved properties such as increased affinity, metabolic stability,membrane permeability or solubility.

In addition, such conformationally constrained peptides are expected topresent the antigenic PCSK9 epitope in a conformation similar to theirnative loop conformation, thereby inducing anti-PCSK9 antibodies moresusceptible to recognize intact, native self PCSK9 molecules or with anincreased affinity to recognize self PCSK9 molecules.

Methods of conformational constraint are well known in the art andinclude, without limitation, bridging and cyclization.

There are several approaches known in the prior art to introduceconformational constraints into a linear peptide. For example, bridgingbetween two neighbouring amino acids in a peptide leads to a localconformational modification, the flexibility of which is limited incomparison with that of regular peptides. Some possibilities for formingsuch bridges include incorporation of lactams and piperazinones (forreview see Giannis and Kolter, Angew. Chem. Int. Ed., 1993, 32: 1244).

As used herein in reference to a peptide, the term “cyclic” refers to astructure including an intramolecular bond between two non-adjacentamino acids or amino acid analogs.

The cyclization can be effected through a covalent or non-covalent bond.Intramolecular bonds include, but are not limited to, backbone tobackbone, side-chain to backbone, side-chain to side-chain, side chainto end-group, end-to-end bonds. Methods of cyclization include, withoutlimitation, formation of an amide bond between the N-term residue andthe C-term residue of a peptide, formation of a disulfide bond betweenthe side-chains of non-adjacent amino acids or amino acid analogs;formation of an amide bond between the side-chains of Lys and Asp/Gluresidues; formation of an ester bond between serine residues and Asp/Gluresidues; formation of a lactam bond, for example, between a side-chaingroup of one amino acid or analog thereof to the N-terminal amine of theamino-terminal residue; and formation of lysinonorleucine and dityrosinebonds. Carbon versions of a disulfide linkage, for example an ethenyl orethyl linkage, could also be used (J. Peptide Sc., 2008, 14, 898-902) aswell as alkylation reactions with an appropriately polysubstitutedelectrophilic reagent such as a di-, tri- or tetrahaloalkane (PNAS,2008, 105(40), 15293-15298; ChemBioChem, 2005, 6, 821-824). Variousmodified proline analogs can also be used to incorporate conformationalconstraints into peptides (Zhang et al., J. Med Chem., 1996, 39:2738-2744; Pfeifer and Robinson, Chem. Comm., 1998, 1977-1978).Chemistries that may be used to cyclise peptides of the invention resultin peptides cyclised with a bond including, but not limiting to thefollowing: lactam, hydrazone, oxime, thiazolidine, thioether orsulfonium bonds.

Yet another approach in the design of conformationally constrainedpeptides, which is described in U.S. Ser. No. 10/114,918, is to attach ashort amino acid sequence of interest to a template, to generate acyclic constrained peptide. Such cyclic peptides are not onlystructurally stabilized by their templates, and thereby offerthree-dimensional conformations that may imitate conformational epitopeson native proteins such as on viruses and parasites or on self proteins(autologous mammalian proteins such as PCSK9), but they are also moreresistant than linear peptides to proteolytic degradation in serum. U.S.Ser. No. 10/114,918 further discloses the synthesis of conformationallyconstrained cross-linked peptides by preparation of synthetic aminoacids for backbone coupling to appropriately positioned amino acids inorder to stabilize the supersecondary structure of peptides.Cross-linking can be achieved by amide coupling of the primary aminogroup of an orthogonally protected (2S, 3R)-3-aminoproline residue to asuitably positioned side chain carboxyl group of glutamate. Thisapproach has been followed in the preparation of conformationallyconstrained tetrapeptide repeats of the CS protein wherein at least oneproline has been replaced by 2S, 3R)-3-aminoproline and, in order tointroduce a side chain carboxyl group, glutamate has been incorporatedas a replacement for alanine.

Cross-linking strategies also include the application of the Grubbsring-closing metathesis reaction to form ‘stapled’ peptides designed tomimic alpha-helical conformations (Angew. Int. Ed. Engl., 1998, 37, 3281JACS, 2000, 122, 5891); use of poly-functionalised saccharides use of atryptathionine linkage (Chemistry Eu. J., 2008, 24, 3404-3409); use of‘click’ reaction of azides and alkynes which could be incorporated aseither a side chain amino acid residues or located within the backboneof the peptide sequence (Drug Disc. Today, 2003, 8(24), 1128-1137). Itis also known in the literature that metal ions can stabiliseconstrained conformations of linear peptides through sequesteringspecific residues e.g. histidine, which co-ordinate to metal cations(Angew. Int. Ed. Engl., 2003, 42, 421). Similarly, functionalising alinear peptide sequence with non-natural acid and amine functionality,or polyamine and polyacid functionality can be used to allow access tocyclised structures following activation and amide bond formation.

According to one embodiment, the antigenic PCSK9 peptide isconformationally constrained by intramolecular covalent bonding of twonon-adjacent amino acids of the antigenic PCSK9 peptide to each other,e.g. the N- and C-terminal amino acids.

According to another embodiment, the antigenic PCSK9 peptide of theinvention is conformationally constrained by covalent binding to ascaffold molecule. According to a further embodiment, the antigenicPCSK9 peptide is simply constrained, i.e. coupled either at one end, (Cor N terminus) or through another amino acid not located at either end,to the scaffold molecule. According to another embodiment, the antigenicPCSK9 peptide is doubly constrained, i.e. coupled at both C and Ntermini to the scaffold molecule. According to another embodiment, theantigenic peptide is constrained by cyclising via the templating effectof a heterochiral Diproline unit (D-Pro-L-Pro) (Spath et al, 1998,Helvetica Chimica Acta 81, p 1726-1738).

The scaffold (also called ‘platform’) can be any molecule which iscapable of reducing, through covalent bonding, the number ofconformations which the antigenic PCSK9 peptide can assume. Examples ofconformation-constraining scaffolds include proteins and peptides, forexample lipocalin-related molecules such as beta-barrel containingthioredoxin and thioredoxin-like proteins, nucleases (e.g. RNaseA),proteases (e.g. trypsin), protease inhibitors (e.g. eglin C), antibodiesor structurally-rigid fragments thereof, fluorescent proteins such asGFP or YFP, conotoxins, loop regions of fibronectin type III domain,CTL-A4, and virus-like particles (VLPs).

Other suitable platform molecules include carbohydrates such assepharose. The platform may be a linear or circular molecule, forexample, closed to form a loop. The platform is generally heterologouswith respect to the antigenic PCSK9 peptide. Such conformationallyconstrained peptides linked to a platform are thought to be moreresistant to proteolytic degradation than linear peptide.

According to a preferred embodiment, the scaffold is an immunogeniccarrier as defined in the present application. In a further embodiment,the antigenic PCSK9 peptide is simply constrained onto the immunogeniccarrier. In another further embodiment, the antigenic PCSK9 peptide isdoubly constrained onto the immunogenic carrier. In this manner, theantigenic PCSK9 peptide forms a conformationally constrained loopstructure which has proven to be a particularly suitable structure as anintracellular recognition molecule.

The antigenic PCSK9 peptides of the invention may be modified for theease of conjugation to a platform, for example by the addition of aterminal cysteine at one or both ends and/or by the addition of a linkersequence, such a double glycine head or tail plus a terminal cysteine, alinker terminating with a lysine residue or any other linker known tothose skilled in the art to perform such function. Details of suchlinkers are disclosed hereafter. Bioorthogonal chemistry (such as theclick reaction described above) to couple the full peptide sequence tothe carrier, thus avoiding any regiochemical and chemoselectivityissues, might also be used. Rigidified linkers such as the one describedin Jones et al. Angew. Chem. Int. Ed. 2002, 41:4241-4244 are known toelicit an improved immunological response and might also be used.

In a further embodiment, the antigenic PCSK9 peptide is attached to amultivalent template, which itself is coupled to the carrier, thusincreasing the density of the antigen (see below). The multivalenttemplate could be an appropriately functionalised polymer or oligomersuch as (but not limited to) oligoglutamate or oligochitosan (see FIG.19).

Immunogenic Carrier of the Invention

In an embodiment of the present invention, the antigenic PCSK9 peptideof the invention is linked to an immunogenic carrier molecule to formimmunogens for vaccination protocols, preferably wherein the carriermolecule is not related to the native PCSK9 molecule.

The term “immunogenic carrier” herein includes those materials whichhave the property of independently eliciting an immunogenic response ina host animal and which can be covalently coupled to a peptide,polypeptide or protein either directly via formation of peptide or esterbonds between free carboxyl, amino or hydroxyl groups in the peptide,polypeptide or protein and corresponding groups on the immunogeniccarrier material, or alternatively by bonding through a conventionalbifunctional linking group, or as a fusion protein.

The types of carriers used in the immunogens of the present inventionwill be readily known to the person skilled in the art. Examples of suchimmunogenic carriers are: serum albumins such as bovine serum albumin(BSA); globulins; thyroglobulins; hemoglobins; hemocyanins (particularlyKeyhole Limpet Hemocyanin [KLH]); polylysin; polyglutamic acid;lysine-glutamic acid copolymers; copolymers containing lysine orornithine; liposome carriers; the purified protein derivative oftuberculin (PPD); inactivated bacterial toxins or toxoids such astetanus or diptheria toxins (TT and DT) or fragment C of TT, CRM197 (anontoxic but antigenically identical variant of diphtheria toxin) otherDT point mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol.Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107and other mutations described by Nicholls and Youle in GeneticallyEngineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion ormutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and othermutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat. No.4,950,740; mutation of at least one or more residues Lys 516, Lys 526,Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat. No.5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S. Pat.No. 5,843,711, pneumococcal pneumolysin (Kuo et al (1995) Infect Immun63; 2706-13) including ply detoxified in some fashion for exampledPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtEare disclosed in WO 00/37105 or WO 00/39299) and fusions of Pht proteinsfor example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334, WO03/54007, WO2009/000826), OMPC (meningococcal outer membraneprotein—usually extracted from N. meningitidis serogroup B—EP0372501),PorB (from N. meningitidis), PD (Haemophilus influenzae protein D—see,e.g., EP 0 594 610 B), or immunologically functional equivalentsthereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins(WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761).

In a preferred embodiment, the immunogenic carrier of the invention isCRM197.

In another embodiment, the immunogenic carrier is a virus-like particle(VLPs), preferably a recombinant virus-like particle.

As used herein, the term “virus-like particle” refers to a structureresembling a virus particle but which has been demonstrated to be nonpathogenic. In general, virus-like particles lack at least part of theviral genome. Also, virus-like particles can often be produced in largequantities by heterologous expression and can be easily purified. Avirus-like particle in accordance with the invention may contain nucleicacid distinct from their genome. A typical and preferred embodiment of avirus-like particle in accordance with the present invention is a viralcapsid such as the viral capsid of the corresponding virus,bacteriophage, or RNA-phage.

As used herein, the term “virus-like particle of a bacteriophage” refersto a virus-like particle resembling the structure of a bacteriophage,being non replicative and noninfectious, and lacking at least the geneor genes encoding for the replication machinery of the bacteriophage,and typically also lacking the gene or genes encoding the protein orproteins responsible for viral attachment to or entry into the host.This definition should, however, also encompass virus-like particles ofbacteriophages, in which the aforementioned gene or genes are stillpresent but inactive, and, therefore, also leading to non-replicativeand noninfectious virus-like particles of a bacteriophage. The capsidstructure formed from the self-assembly of 180 subunits of RNA phagecoat protein and optionally containing host RNA is herein referred to asa “VLP of RNA phage coat protein”. Specific examples are the VLP ofQbeta, MS2, PP7 or AP205 coat proteins. In the specific case of Qbetacoat protein, for example, the VLP may either be assembled exclusivelyfrom Qbeta CP subunits (generated by expression of a Qbeta CP genecontaining, for example, a TAA stop codon precluding any expression ofthe longer A1 protein through suppression, see Kozlovska, T. M., et al.,Intervirology 39: 9-15 (1996)), or additionally contain A1 proteinsubunits in the capsid assembly. Generally, the percentage of Qbeta A1protein relative to Qbeta CP in the capsid assembly will be limited, inorder to ensure capsid formation.

Examples of VLPs suitable as immunogenic carriers in the context of thepresent invention include, but are not limited to, VLPs of Qbeta, MS2,PP7, AP205 and other bacteriophage coat proteins, the capsid and coreproteins of Hepatitis B virus (Ulrich, et al., Virus Res. 50: 141-182(1998)), measles virus (Warnes, et al., Gene 160: 173-178 (1995)),Sindbis virus, rotavirus (U.S. Pat. Nos. 5,071,651 and 5,374,426),foot-and-mouth-disease virus (Twomey, et al., Vaccine 13: 1603-1610,(1995)), Norwalk virus (Jiang, X., et al., Science 250: 1580-1583(1990); Matsui, S. M., et al., J Clin. Invest. 87: 1456-1461 (1991)),the retroviral GAG protein (PCT Patent Appl. No. WO 96/30523), theretrotransposon Ty protein pl, the surface protein of Hepatitis B virus(WO 92/11291), human papilloma virus (WO 98/15631), human polyoma virus(Sasnauskas K., et al., Biol. Chem. 380 (3): 381-386 (1999); SasnauskasK., et al., Generation of recombinant virus-like particles of differentpolyomaviruses in yeast. 3rd Interational Workshop “Virus-like particlesas vaccines.” Berlin, September 26-29 (2001)), RNA phages, Ty, frphage,GA-phage, AP 205-phage and, in particular, Qbeta-phage, Cowpea chloroticmottle virus, cowpea mosaic virus, human papilloma viruses (HPV), bovinepapilloma viruses, porcine parvovirus, parvoviruses such as B19, porcine(PPV) and canine (CPV) parvovirues, caliciviruses (e.g. Norwalk virus,rabbit hemorrhagic disease virus [RHDV]), animal hepadnavirus coreAntigen VLPs, filamentous/rod-shaped plant viruses, including but notlimited to Tobacco Mosaic Virus (TMV), Potato Virus X (PVX), PapayaMosaic Virus (PapMV), Alfalfa Mosaic Virus (AIMV), and Johnson GrassMosaic Virus (JGMV), insect viruses such as flock house virus (FHV) andtetraviruses, polyomaviruses such as Murine Polyomavirus (MPyV), MurinePneumotropic Virus (MPtV), BK virus (BKV), and JC virus (JCV).

As will be readily apparent to those skilled in the art, the VLP to beused as an immunogenic carrier of the invention is not limited to anyspecific form. The particle can be synthesized chemically or through abiological process, which can be natural or nonnatural. By way ofexample, this type of embodiment includes a virus-like particle or arecombinant form thereof. In a more specific embodiment, the VLP cancomprise, or alternatively consist of, recombinant polypeptides of anyof the virus known to form a VLP. The virus-like particle can furthercomprise, or alternatively consist of, one or more fragments of suchpolypeptides, as well as variants of such polypeptides. Variants ofpolypeptides can share, for example, at least 80%, 85%, 90%, 95%, 97%,or 99% identity at the amino acid level with their wild-typecounterparts. Variant VLPs suitable for use in the present invention canbe derived from any organism so long as they are able to form a“virus-like particle” and can be used as an “immunogenic carrier” asdefined herein.

Preferred VLPs according to the invention include the capsid protein orsurface antigen of HBV (HBcAg and HBsAg respectively) or recombinantproteins or fragments thereof, and the coat proteins of RNA-phages orrecombinant proteins or fragments thereof, more preferably the coatprotein of Qbeta or recombinant proteins or fragments thereof. In oneembodiment, the immunogic carrier used in combination with an antigenicPCSK9 peptide of the invention is an HBcAg protein. Examples of HBcAgproteins that can be used in the context of the present invention can bereadily determined by one skilled in the art. Examples include, but arelimited to, HBV core proteins described in Yuan et al., (J. Virol. 73:10122-10128 (1999)), and in WO00/198333, WO 00/177158, WO 00/214478, WOWO00/32227, WO01/85208, WO02/056905, WO03/024480, and WO03/024481.HBcAgs suitable for use in the present invention can be derived from anyorganism so long as they are able to form a “virus-like particle” andcan be used as an “immunogenic carrier” as defined herein.

HBcAg variants of particular interest that could be used in the contextof the present invention are those variants in which one or morenaturally resident cysteine residues have been either deleted orsubstituted. It is well known in the art that free cysteine residues canbe involved in a number of chemical side reactions including disulfideexchanges, reaction with chemical substances or metabolites that are,for example, injected or formed in a combination therapy with othersubstances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. The toxic adducts would thus be distributed between amultiplicity of species, which individually may each be present at lowconcentration, but reach toxic levels when together. In view of theabove, one advantage to the use of HBcAgs in vaccine compositions whichhave been modified to remove naturally resident cysteine residues isthat sites to which toxic species can bind when antigens or antigenicdeterminants are attached would be reduced in number or eliminatedaltogether.

In addition, the processed form of HBcAg lacking the N-terminal leadersequence of the Hepatitis B core antigen precursor protein can also beused in the context of the invention, especially when HBcAg is producedunder conditions where processing will not occur (e.g. expression inbacterial systems).

Other HBcAg variants according to the invention include i) polypeptidesequence having at least 80%, 85%, 90%, 95%, 97% or 99% identical to oneof the wild-type HBcAg amino acid sequences, or a subportion thereof,using conventionally using known computer programs, ii) C-terminaltruncation mutants including mutants where 1, 5, 10, 15, 20, 25, 30, 34or 35, amino acids have been removed from the C-terminus, ii) N-terminaltruncation mutants including mutants where 1, 2, 5, 7, 9, 10, 12, 14,15, or 17 amino acids have been removed from the N-terminus, iii)mutants truncated in both N-terminal and C-terminal include HBcAgs where1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed fromthe N-terminus and 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids havebeen removed from the C-terminus.

Still other HBcAg variant proteins within the scope of the invention arethose variants modified in order to enhance immunogenic presentation ofa foreign epitope wherein one or more of the four arginine repeats hasbeen deleted, but in which the C-terminal cysteine is retained (see e.g.WO01/98333), and chimeric C-terminally truncated HBcAg such as thosedescribed in WO02/14478, WO03/102165 and WO04/053091.

In another embodiment, the immunogic carrier used in combination with anantigenic PCSK9 peptide of the invention is an HBsAg protein. HBsAgproteins that could be used in the context of the present invention canbe readily determined by one skilled in the art. Examples include, butare limited to, HBV surface proteins described in U.S. Pat. No.5,792,463, WO02/10416, and WO08/020331. HBsAgs suitable for use in thepresent invention can be derived from any organism so long as they areable to form a “virus-like particle” and can be used as an “immunogeniccarrier” as defined herein.

In still another embodiment, the immunogic carrier used in combinationwith an antigenic PCSK9 peptide or polypeptide of the invention is aQbeta coat protein.

Qbeta coat protein was found to self-assemble into capsids whenexpressed in E. coli (Kozlovska™. et al., GENE 137: 133-137 (1993)). Theobtained capsids or virus-like particles showed an icosahedralphage-like capsid structure with a diameter of 25 nm and T=3 quasisymmetry. Further, the crystal structure of phage Qss has been solved.The capsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4: 5435554 (1996)) leading to a remarkable stabilityof the capsid of Qbeta coat protein. Qbeta capsid protein also showsunusual resistance to organic solvents and denaturing agents. The highstability of the capsid of Qbeta coat protein is an advantageousfeature, in particular, for its use in immunization and vaccination ofmammals and humans in accordance of the present invention.

Examples of Qbeta coat proteins that can be used in the context of thepresent invention can be readily determined by one skilled in the art.Examples have been extensively described in WO02/056905, WO03/024480,WO03/024481 (incorporated by reference in their entirety) and include,but are not limited to, amino acid sequences disclosed in PIR database,accession No. VCBPQbeta referring to Qbeta CP; Accession No. AAA16663referring to Qbeta A1 protein; and variants thereof including variantsproteins in which the N-terminal methionine is cleaved; C-terminaltruncated forms of Qbeta A1 missing as much as 100, 150 or 180 aminoacids; variant proteins which have been modified by the removal of alysine residue by deletion or substitution or by the addition of alysine residue by substitution or insertion (see for example Qbeta-240,Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259 disclosed in WO03/024481,incorporated by reference in its entirety), and variants exhibiting atleast 80%, 85%, 90%, 95%, 97%, or 99% identity to any of the Qbeta coreproteins described above. Variant Qbeta coat proteins suitable for usein the present invention can be derived from any organism so long asthey are able to form a “virus-like particle” and can be used as“immunogenic carriers” as defined herein.

The antigenic PCSK9 peptides of the invention may be coupled toimmunogenic carriers via chemical conjugation or by expression ofgenetically engineered fusion partners. The coupling does notnecessarily need to be direct, but can occur through linker sequences.More generally, in the case that antigenic peptides either fused,conjugated or otherwise attached to an immunogenic carrier, spacer orlinker sequences are typically added at one or both ends of theantigenic peptides. Such linker sequences generally comprise sequencesrecognized by the proteasome, proteases of the endosomes or othervesicular compartment of the cell.

In one embodiment, the peptides of the present invention are expressedas fusion proteins with the immunogenic carrier. Fusion of the peptidecan be effected by insertion into the immunogenic carrier primarysequence, or by fusion to either the N- or C-terminus of the immunogeniccarrier. Hereinafter, when referring to fusion proteins of a peptide toan immunogenic carrier, the fusion to either ends of the subunitsequence or internal insertion of the peptide within the carriersequence are encompassed. Fusion, as referred to hereinafter, may beeffected by insertion of the antigenic peptide into the sequence ofcarrier, by substitution of part of the sequence of the carrier with theantigenic peptide, or by a combination of deletion, substitution orinsertions.

When the immunogenic carrier is a VLP, the chimeric antigenicpeptide-VLP subunit will be in general capable of self-assembly into aVLP. VLP displaying epitopes fused to their subunits are also hereinreferred to as chimeric VLPs. For example, EP 0 421 635 B describes theuse of chimaeric hepadnavirus core antigen particles to present foreignpeptide sequences in a virus-like particle.

Flanking amino acid residues may be added to either end of the sequenceof the antigenic peptide to be fused to either end of the sequence ofthe subunit of a VLP, or for internal insertion of such peptidicsequence into the sequence of the subunit of a VLP. Glycine and serineresidues are particularly favored amino acids to be used in the flankingsequences added to the peptide to be fused. Glycine residues conferadditional flexibility, which may diminish the potentially destabilizingeffect of fusing a foreign sequence into the sequence of a VLP subunit.

In a specific embodiment of the invention, the immunogenic carrier is aHBcAg VLP. Fusion proteins of the antigenic peptide to either theN-terminus of a HBcAg (Neyrinck, S. et al., Nature Med. 5: 11571163(1999)) or insertions in the so called major immunodominant region (MIR)have been described (Pumpens, P. and Grens, E., Intervirology 44: 98114(2001)), WO 01/98333), and are specific embodiments of the invention.Naturally occurring variants of HBcAg with deletions in the MIR havealso been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114(2001)), and fusions to the N- or C-terminus, as well as insertions atthe position of the MIR corresponding to the site of deletion ascompared to a wt HBcAg are further embodiments of the invention. Fusionsto the C-terminus have also been described (Pumpens, P. and Grens, E.,Intervirology 44: 98-114 (2001)). One skilled in the art will easilyfind guidance on how to construct fusion proteins using classicalmolecular biology techniques. Vectors and plasmids encoding HBcAg andHBcAg fusion proteins and useful for the expression of a HBcAg and HBcAgfusion proteins have been described (Pumpens, P. and #38; Grens, E.Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) and can be used in the practice of the invention. Animportant factor for the optimization of the efficiency of self-assemblyand of the display of the epitope to be inserted in the MIR of HBcAg isthe choice of the insertion site, as well as the number of amino acidsto be deleted from the HBcAg sequence within the MIR (Pumpens, P. andGrens, E., Intervirology 44: 98-114 (2001); EP 0 421 635; U.S. Pat. No.6,231,864) upon insertion, or in other words, which amino acids formHBcAg are to be substituted with the new epitope. For example,substitution of HBcAg amino acids 76-80, 79-81, 79-80, 75-85 or 80-81with foreign epitopes has been described (Pumpens, P. and Grens, E.,Intervirology 44: 98-114 (2001); EP0421635; U.S. Pat. No. 6,231,864,WO00/26385). HBcAg contains a long arginine tail (Pumpens, P. and Grens,E., Intervirology 44: 98-114 (2001)) which is dispensable for capsidassembly and capable of binding nucleic acids (Pumpens, P. and Grens,E., Intervirology 44: 98-114 (2001)). HBcAg either comprising or lackingthis arginine tail are both embodiments of the invention.

In another specific embodiment of the invention, the immunogenic carrieris a VLP of a RNA phage, preferably Qbeta. The major coat proteins ofRNA phages spontaneously assemble into VLPs upon expression in bacteria,and in particular in E. coli. Fusion protein constructs whereinantigenic peptides have been fused to the C-terminus of a truncated formof the A1 protein of Qbeta, or inserted within the A1 protein have beendescribed (Kozlovska, T. M., et al., Intervirology, 39: 9-15 (1996)).The A1 protein is generated by suppression at the UGA stop codon and hasa length of 329 aa, or 328 aa, if the cleavage of the N-terminalmethionine is taken into account. Cleavage of the N-terminal methioninebefore an alanine (the second amino acid encoded by the Qbeta CP gene)usually takes place in E. coli, and such is the case for N-termini ofthe Qbeta coat proteins. The part of the A1 gene, 3′ of the UGA ambercodon encodes the CP extension, which has a length of 195 amino acids.Insertion of the antigenic peptide between position 72 and 73 of the CPextension leads to further embodiments of the invention (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). Fusion of an antigenicpeptide at the C-terminus of a C-terminally truncated Qbeta A1 proteinleads to further preferred embodiments of the invention. For example,Kozlovska et al., (Intervirology, 39: 9-15 (1996)) describe Qbeta A1protein fusions where the epitope is fused at the C-terminus of theQbeta CP extension truncated at position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)),assembly of the particles displaying the fused epitopes typicallyrequires the presence of both the A1 protein-antigen fusion and the wtCP to form a mosaic particle. However, embodiments comprising virus-likeparticles, and hereby in particular the VLPs of the RNA phage Qbeta coatprotein, which are exclusively composed of VLP subunits having anantigenic peptide fused thereto, are also within the scope of thepresent invention.

The production of mosaic particles may be effected in a number of ways.Kozlovska et al., Intervirology, 39: 9-15 (1996), describe threemethods, which all can be used in the practice of the invention. In thefirst approach, efficient display of the fused epitope on the VLPs ismediated by the expression of the plasmid encoding the Qbeta A1l proteinfusion having a UGA stop codon between CP and CP extension in a E. colistrain harboring a plasmid encoding a cloned UGA suppressor tRNA whichleads to translation of the UGA codon into Trp (pISM3001 plasmid (SmileyB. K., et al., Gene 134: 33-40 (1993))). In another approach, the CPgene stop codon is modified into UAA, and a second plasmid expressingthe A1 protein-antigen fusion is cotransformed. The second plasmidencodes a different antibiotic resistance and the origin of replicationis compatible with the first plasmid. In a third approach, CP and the A1protein-antigen fusion are encoded in a bicistronic manner, operativelylinked to a promoter such as the Trp promoter, as described in FIG. 1 ofKozlovska et al., Intervirology, 39: 9-15 (1996). Further VLPs suitablefor fusion of antigens or antigenic determinants are described inWO03/024481 and include bacteriophage fr, RNA phase MS-2, capsid proteinof papillomavirus, retrotransposon Ty, yeast and alsoRetrovirus-like-particles, HIV2 Gag, Cowpea Mosaic Virus, parvovirus VP2VLP, HBsAg (U.S. Pat. No. 4,722,840, EP0020416B1). Examples of chimericVLPs suitable for the practice of the invention are also those describedin Intervirology 39: 1 (1996). Further examples of VLPs contemplated foruse in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33,HPV-45, CRPV, COPV, HIV GAG, Tobacco Mosaic Virus. Virus-like particlesof SV-40, Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus andNorwalk virus.

For any recombinantly expressed antigenic PCSK9 peptide according to theinvention coupled or not to an immunogenic carrier, the nucleic acidwhich encodes said peptide or protein also forms an aspect of thepresent invention, as does an expression vector comprising the nucleicacid, and a host cell containing the expression vector (autonomously orchromosomally inserted). A method of recombinantly producing the peptideor protein by expressing it in the above host cell and isolating theimmunogen therefrom is a further aspect of the invention. Thefull-length native PCSK9 molecule or the full-length native DNA sequenceencoding it are not covered by the present invention.

In another embodiment, the peptide of the invention is chemicallycoupled to an immunogenic carrier, using techniques well known in theart. Conjugation can occur to allow free movement of peptides via singlepoint conjugation (e.g. either N-terminal or C-terminal point) or aslocked down structure where both ends of peptides are conjugated toeither a immunogenic carrier protein or to a scaffold structure such asa VLP. Conjugation occurs via conjugation chemistry known to thoseskilled in the art such as via cysteine residues, lysine residues orother carboxy moiety's commonly known as conjugation points such asglutamic acid or aspartic acid. Thus, for example, for direct covalentcoupling it is possible to utilise a carbodiimide, glutaraldehyde or(N-[y-malcimidobutyryloxy] succinimide ester, utilising commoncommercially available heterobifunctional linkers such as CDAP and SPDP(using manufacturers instructions). Examples of conjugation of peptides,particularly cyclised peptides, to a protein carrier via acylhydrazinepeptide derivatives are described in WO03/092714. After the couplingreaction, the immunogen can easily be isolated and purified by means ofa dialysis method, a gel filtration method, a fractionation method etc.Peptides terminating with a cysteine residue (preferably with a linkeroutside the cyclised region) may be conveniently conjugated to a carrierprotein via maleimide chemistry.

When the immunogenic carrier is a VLP, several antigenic peptide, eitherhaving an identical amino acid sequence or a different amino acidsequence, may be coupled to a single VLP molecule, leading preferably toa repetitive and ordered structure presenting several antigenicdeterminants in an oriented manner as described in WO00/32227,WO03/024481, WO02/056905 and WO04/007538.

In a preferred embodiment, the antigenic PCSK9 peptide is bound to theVLP by way of chemical cross-linking, typically and preferably by usinga heterobifunctional cross-linker. Several hetero-bifunctionalcross-linkers are known to the art. In some embodiments, thehetero-bifunctional crosslinker contains a functional group which canreact with first attachment sites, i. e. with the side-chain amino groupof lysine residues of the VLP or VLP subunit, and a further functionalgroup which can react with a preferred second attachment site, i. e. acysteine residue fused to the antigenic peptide and optionally also madeavailable for reaction by reduction. The first step of the procedure,typically called the derivatization, is the reaction of the VLP with thecross-linker. The product of this reaction is an activated VLP, alsocalled activated carrier. In the second step, unreacted cross-linker isremoved using usual methods such as gel filtration or dialysis. In thethird step, the antigenic peptide is reacted with the activated VLP, andthis step is typically called the coupling step. Unreacted antigenicpeptide may be optionally removed in a fourth step, for example bydialysis. Several hetero-bifunctional crosslinkers are known to the art.These include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS,Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIAand other cross-linkers available for example from the Pierce ChemicalCompany (Rockford, Ill., USA), and having one functional group reactivetowards amino groups and one functional group reactive towards cysteineresidues. The above mentioned cross-linkers all lead to formation of athioether linkage.

Another class of cross-linkers suitable in the practice of the inventionis characterized by the introduction of a disulfide linkage between theantigenic peptide and the VLP upon coupling. Preferred cross-linkersbelonging to this class include for example SPDP and Sulfo-LC-SPDP(Pierce). The extent of derivatization of the VLP with cross-linker canbe influenced by varying experimental conditions such as theconcentration of each of the reaction partners, the excess of onereagent over the other, the pH, the temperature and the ionic strength.The degree of coupling, i. e. the amount of antigenic peptide persubunits of the VLP can be adjusted by varying the experimentalconditions described above to match the requirements of the vaccine.

Another method of binding of antigenic peptides to the VLP, is thelinking of a lysine residue on the surface of the VLP with a cysteineresidue on the antigenic peptide. In some embodiments, fusion of anamino acid linker containing a cysteine residue, as a second attachmentsite or as a part thereof, to the antigenic peptide for coupling to theVLP may be required. In general, flexible amino acid linkers arefavored. Examples of the amino acid linker are selected from the groupconsisting of: (a) CGG; (b) N-terminal gamma 1-linker; (c) N-terminalgamma 3-linker; (d) Ig hinge regions; (e) N-terminal glycine linkers;(f) (G) kC (G) n with n=0-12 and k=0-5; (g) N-terminal glycine-serinelinkers; (h) (G) kC (G) m (S) i (GGGGS) n with n=0-3, k=0-5, m=0-10,i=0-2; (i) GGC; (k) GGC-NH2; (l) C-terminal gamma 1-linker; (m)C-terminal gamma 3-linker; (n) C-terminal glycine linkers; (o) (G) nC(G) k with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q)(G) m (S) t (GGGGS) n (G) oC (G) k with n=0-3, k=0-5, m=0-10, 1=0-2, ando=0-8. Further examples of amino acid linkers are the hinge region ofimmunoglobulins, glycine serine linkers (GGGGS) n, and glycine linkers(G) n all further containing a cysteine residue as second attachmentsite and optionally further glycine residues. Typically preferredexamples of said amino acid linkers are N-terminal gamma 1: CGDKTHTSPP(SEQ ID NO:608); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:609);N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO:610); C-terminal gamma3: PKPSTPPGSSGGAPGGCG (SEQ ID NO:611); N-terminal glycine linker: GCGGGG(SEQ ID NO:612) and C-terminal glycine linker: GGGGCG (SEQ ID NO:613).Other amino acid linkers particularly suitable in the practice of theinvention, when a hydrophobic antigenic peptide is bound to a VLP, areCGKKGG (SEQ ID NO:614), or CGDEGG (SEQ ID NO:615) for N-terminallinkers, or GGKKGC (SEQ ID NO:616 and GGEDGC (SEQ ID NO:617), for theC-terminal linkers. For the C-terminal linkers, the terminal cysteine isoptionally C-terminally amidated.

In some embodiments of the present invention, GGCG (SEQ ID NO:618), GGCor GGC-NH2 (“NH2” stands for amidation) linkers at the C-terminus of thepeptide or CGG at its N-terminus are preferred as amino acid linkers. Ingeneral, glycine residues will be inserted between bulky amino acids andthe cysteine to be used as second attachment site, to avoid potentialsteric hindrance of the bulkier amino acid in the coupling reaction. Ina further embodiment of the invention, the amino acid linker GGC-NH2 isfused to the C-terminus of the antigenic peptide.

The cysteine residue present on the antigenic peptide has to be in itsreduced state to react with the hetero-bifunctional cross-linker on theactivated VLP, that is a free cysteine or a cysteine residue with a freesulfhydryl group has to be available. In the instance where the cysteineresidue to function as binding site is in an oxidized form, for exampleif it is forming a disulfide bridge, reduction of this disulfide bridgewith e. g. DTT, TCEP or p-mercaptoethanol is required. Lowconcentrations of reducing agent are compatible with coupling asdescribed in WO 02/05690, higher concentrations inhibit the couplingreaction, as a skilled artisan would know, in which case the reductandhas to be removed or its concentration decreased prior to coupling, e.g. by dialysis, gel filtration or reverse phase HPLC.

Binding of the antigenic peptide to the VLP by using ahetero-bifunctional cross-linker according to the methods describedabove, allows coupling of the antigenic peptide to the VLP in anoriented fashion. Other methods of binding the antigenic peptide to theVLP include methods wherein the antigenic peptide is cross-linked to theVLP using the carbodiimide EDC, and NHS.

In other methods, the antigenic peptide is attached to the VLP using ahomo-bifunctional cross-linker such as glutaraldehyde, DSGBM [PEO] 4,BS3, (Pierce Chemical Company, Rockford, Ill., USA) or other knownhomo-bifunctional cross-linkers with functional groups reactive towardsamine groups or carboxyl groups of the VLP. Other methods of binding theVLP to an antigenic peptide include methods where the VLP isbiotinylated, and the antigenic peptide expressed as astreptavidin-fusion protein, or methods wherein both the antigenicpeptide and the VLP are biotinylated, for example as described in WO00/23955. In this case, the antigenic peptide may be first bound tostreptavidin or avidin by adjusting the ratio of antigenic peptide tostreptavidin such that free binding sites are still available forbinding of the VLP, which is added in the next step. Alternatively, allcomponents may be mixed in a “one pot” reaction. Other ligand-receptorpairs, where a soluble form of the receptor and of the ligand isavailable, and are capable of being cross-linked to the VLP or theantigenic peptide, may be used as binding agents for binding antigenicpeptide to the VLP. Alternatively, either the ligand or the receptor maybe fused to the antigenic peptide, and so mediate binding to the VLPchemically bound or fused either to the receptor, or the ligandrespectively. Fusion may also be effected by insertion or substitution.

One or several antigen molecules can be attached to one subunit of thecapsid or VLP of RNA phages coat proteins, preferably through theexposed lysine residues of the VLP of RNA phages, if stericallyallowable. A specific feature of the VLP of the coat protein of RNAphages and in particular of the QP coat protein VLP is thus thepossibility to couple several antigens per subunit. This allows for thegeneration of a dense antigen array. VLPs or capsids of Q coat proteindisplay a defined number of lysine residues on their surface, with adefined topology with three lysine residues pointing towards theinterior of the capsid and interacting with the RNA, and four otherlysine residues exposed to the exterior of the capsid. These definedproperties favor the attachment of antigens to the exterior of theparticle, rather than to the interior of the particle where the lysineresidues interact with RNA. VLPs of other RNA phage coat proteins alsohave a defined number of lysine residues on their surface and a definedtopology of these lysine residues.

In a further embodiment of the present invention, the first attachmentsite is a lysine residue and/or the second attachment comprisessulfhydryl group or a cysteine residue. In an even further embodiment ofthe present invention, the first attachment site is a lysine residue andthe second attachment is a cysteine residue. In further embodiments ofthe invention, the antigen or antigenic determinant is bound via acysteine residue, to lysine residues of the VLP of RNA phage coatprotein, and in particular to the VLP of Qbeta coat protein.

Another advantage of the VLPs derived from RNA phages is their highexpression yield in bacteria that allows production of large quantitiesof material at affordable cost. Moreover, the use of the VLPs ascarriers allow the formation of robust antigen arrays and conjugates,respectively, with variable antigen density. In particular, the use ofVLPs of RNA phages, and hereby in particular the use of the VLP of RNAphage Qbeta coat protein allows a very high epitope density to beachieved.

According to an embodiment of the present invention the antigenic PCSK9peptide disclosed herein are linked, preferably chemically cross linked,to CRM197, either directly or via one of the peptide linker disclosedherein, to generate an immunogen. In an embodiment, the antigenic PCSK9peptide disclosed herein is linked to CRM197, by way of chemicalcross-linking as described herein and preferably by using aheterobifunctional cross-linker, as disclosed above.

Preferred heterobifunctional cross-linkers for use with CRM197 are BAANS(bromoacetic acid N-hydroxysuccinimide ester), SMPH(Succinimidyl-6-[β-maleimidopropionamido]hexanoate), Sulfo-MBS,Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIAand other cross-linkers available for example from the Pierce ChemicalCompany (Rockford, Ill., USA). In a preferred embodiment of the presentinvention, the hetero-bifunctional crosslinker is BAANS or SMPH.

Alternatively, cross-linkers suitable allowing the introduction of adisulfide linkage between the antigenic peptide and CRM197 could also beused in the context of the invention. Preferred cross-linkers belongingto this class include for example SPDP and Sulfo-LC-SPDP (Pierce).

In a particular embodiment, when the sequence of the antigenic PCSK9peptide disclosed herein comprises a cysteine, said antigenic PCSK9peptide may be covalently linked to CRM197 directly via said cysteine.

In some embodiments of the invention, immunogenic compositions of theinvention may comprise mixtures of immunogenic conjugates, i.e.immunogenic carriers coupled to one or several antigenic PCSK9 peptidesof the invention. Thus, these immunogenic compositions may be composedof immunogenic carriers which differ in amino acid sequence. Forexample, vaccine compositions could be prepared comprising a “wild-type”VLP and a modified VLP protein in which one or more amino acid residueshave been altered (e. g., deleted, inserted or substituted).Alternatively, the same immunogenic carrier might be used but coupled toantigenic PCSK9 peptides of different amino acid sequences.

The invention therefore also relates to method for producing animmunogen according to the invention comprising i) providing anantigenic PCSK9 peptide according to the invention, ii) providing animmunogenic carrier according to the invention, preferably a VLP, andiii) combining said antigenic PCSK9 peptide and said immunogeniccarrier. In one embodiment, said combining step occurs through chemicalcross-linking, preferably through an heterobifunctional cross-linker.

In an embodiment of the present invention, the antigenic PCSK9 peptidedisclosed herein is linked to an immunogenic carrier molecule. In anembodiment said immunogenic carrier is selected from the groupconsisting of any of the immunogenic carrier described herein. Inanother embodiment said immunogenic carrier is selected from the groupconsisting of: serum albumins such as bovine serum albumin (BSA);globulins; thyroglobulins; hemoglobins; hemocyanins (particularlyKeyhole Limpet Hemocyanin [KLH]) and virus-like particle (VLPs). In apreferred embodiment said immunogenic carrier is Diphtheria Toxoid,CRM197 mutant of diphtheria toxin, Tetanus Toxoid, Keyhole LimpetHemocyanin or virus-like particle (VLPs). In an even preferredembodiment, said immunogenic carrier is DT, CRM197 or a VLP selectedfrom the group consisting of HBcAg VLP, HBsAg VLP, Qbeta VLP, PP7 VLP,PPV VLP, Norwalk Virus VLP or any variant disclosed herein. In an evenpreferred embodiment, said immunogenic carrier is a bacteriophage VLPsuch as Qbeta VLP selected from the group consisting of Qbeta CP; QbetaA1, Qbeta-240, Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259 (disclosedin WO03/024481) or PP7.

In another preferred embodiment, said immunogenic carrier is CRM197.

In an embodiment, said immunogenic carrier is covalently linked to theantigenic PCSK9 peptide disclosed herein either directly or via alinker. In an embodiment, said immunogenic carrier is linked to theantigenic PCSK9 peptide disclosed herein by expression of a fusionprotein as described herein. In another embodiment, the antigenic PCSK9peptide disclosed herein is linked to the immunogenic carrier,preferably a VLP, by way of chemical cross-linking as described hereinand preferably by using a heterobifunctional cross-linker. Severalhetero-bifunctional cross-linkers are known to the art. In someembodiments, the hetero-bifunctional crosslinker contains a functionalgroup which can react with first attachment sites, i.e. with theside-chain amino group of lysine residues of the VLP or VLP subunit, anda further functional group which can react with a preferred secondattachment site, i.e. a cysteine residue fused to the antigenic peptidemade available for reaction by reduction.

Antigenic PCSK9 Peptide of the Invention Comprising a Linker

In an embodiment of the present invention the antigenic PCSK9 peptidedisclosed herein further comprise either at its N-terminus, or at itsC-terminus or at both the N-terminus and C-terminus a linker which isable to react with an attachment site of the immunogenic carrier in achemical cross-linking reaction. In an embodiment, the antigenic PCSK9peptide disclosed herein further comprise at its C-terminus a linkerhaving the formula (G)_(n)C, (G)_(n)SC or (G)_(n)K, preferably (G)_(n)Cwherein n is an integer chosen in the group consisting of 0, 1, 2, 3, 4,5, 6, 7, 8, 9 and 10, preferably in the group consisting of 0, 1, 2, 3,4 and 5, more preferably in the groups consisting of 0, 1, 2 and 3, mostpreferably n is 0 or 1 (where n is equal to 0 said formula represents acysteine). Preferably the antigenic PCSK9 peptide disclosed hereinfurther comprise at its C-terminus a linker having the formula GGGC (SEQID NO:619), GGC, GC or C. In another embodiment of the present inventionthe antigenic PCSK9 peptide disclosed herein further comprise at itsN-terminus a linker having the formula C(G)_(n), CS(G)_(n) or K(G)_(n),preferably C(G)_(n) wherein n is an integer chosen in the groupconsisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably in thegroup consisting of 0, 1, 2, 3, 4 and 5, more preferably in the groupsconsisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (where n isequal to 0, the formula represents a cysteine). Preferably the antigenicPCSK9 peptide disclosed herein further comprise at its N-terminus alinker having the formula CGGG (SEQ ID NO:620), CGG, CG or C.

In another embodiment the antigenic PCSK9 peptide disclosed hereinfurther comprise at its C-terminus a linker having the formula (G)_(n)C,(G)_(n)SC or (G)_(n)K, preferably (G)_(n)C wherein n is an integerchosen in the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,preferably in the group consisting of 0, 1, 2, 3, 4 and 5, morepreferably in the groups consisting of 0, 1, 2 and 3, most preferably n0 or 1 (where n is equal to 0 said formula represents a cysteine) and atits N-terminus a linker having the formula C(G)_(n), CS(G)_(n) orK(G)_(n), preferably C(G)_(n) wherein n is an integer chosen in thegroup consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably inthe group consisting of 0, 1, 2, 3, 4 and 5, more preferably in thegroups consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (where nis equal to 0, the formula represents a cysteine). Preferably theantigenic PCSK9 peptide disclosed herein further comprise at itsN-terminus a linker having the formula CGGG (SEQ ID NO:620), CGG, CG orC and at its C-terminus a linker having the formula GGGC (SEQ IDNO:619), GGC, GC or C. Preferably, the antigenic PCSK9 peptide disclosedherein further comprises at its N-terminus a cysteine and at itsC-terminus a cysteine.

Representative of said antigenic PCSK9 peptides further comprising sucha linker are disclosed at SEQ ID NO 313, 314, 315, 316, 317, 322, 323,324, 325, 326, 327, 328, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418 and 419.

Representative of said antigenic PCSK9 peptides further comprising sucha linker are disclosed at SEQ ID NO 313, 314, 315, 316, 317, 322, 323,324, 325, 326, 327 and 328. Preferred antigenic PCSK9 peptidescomprising a linker are disclosed at SEQ ID Nos 317, 322, 323, 324, 401,402, 403, 413, 414, 415 and 416.

Preferred antigenic PCSK9 peptides comprising a linker are disclosed atSEQ ID Nos 317, 322, 323 and 324.

Most preferred antigenic PCSK9 peptides comprising a linker aredisclosed at SEQ ID Nos 317, 322, 402 and 413.

In one embodiment, the antigenic PCSK9 peptide is cyclised. In oneembodiment, the cyclised antigenic PCSK9 peptide is attached to animmunogenic carrier. In one embodiment, said cyclised antigenic PCSK9peptide is attached to an immunogenic carrier by covalent binding. Inone embodiment, said cyclised antigenic PCSK9 peptide is attached to animmunogenic carrier by covalent binding of one of the side chain of itsamino acids to the carrier. In one embodiment, a cysteine, a GC or a CCfragment comprising a variable number of glycine residues and onecysteine residue is added to the cyclised PCSK9 peptides to enable thecovalent binding to the immunogenic carrier through the added cysteine.

In one embodiment, the antigenic PCSK9 peptide is cyclised and comprisesa cysteine, a (G)_(n)C or a C(G)_(n) fragment wherein n is an integerchosen in the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,preferably in the group consisting of 0, 1, 2, 3, 4 and 5, morepreferably in the groups consisting of 0, 1, 2 and 3, most preferably nis 0 or 1 (where n is equal to 0, the formula represents a cysteine).

Non limitative examples of such conformationally constrained antigenicPCSK9 peptide are the peptides of SEQ ID Nos 318, 319, 320 and 321. Apreferred cyclised peptide is the peptide of SEQ ID Nos 318.

Examples of conjugations of antigenic PCSK9 peptides with carrier orscaffolds described above, all within the scope of the present inventionand constituting various embodiments, using various linkers are providedbelow: Peptide-GGGGGC (SEQ ID NO:621)-scaffold, peptide-GGGGC (SEQ IDNO:622)-scaffold, peptide-GGGC (SEQ ID NO:619)-scaffold,peptide-GGC-scaffold, peptide-GC-scaffold, peptide-C-scaffold,peptide-GGGGGK (SEQ ID NO:624)-scaffold, peptide-GGGGK (SEQ IDNO:625)-scaffold, Peptide-GGGK (SEQ ID NO:626)-scaffold,Peptide-GGK-scaffold, Peptide-GK-scaffold, Peptide-K-scaffold,Peptide-GGGGSC (SEQ ID NO:627)-scaffold, Peptide-GGGSC (SEQ IDNO:628)-scaffold, Peptide-GGSC (SEQ ID NO:629)-scaffold,Peptide-GSC-scaffold, Peptide-SC-scaffold, Scaffold-CSGGGG (SEQ IDNO:630)-Peptide, Scaffold-CSGGG (SEQ ID NO:631)-Peptide, Scaffold-CSGG(SEQ ID NO:632)-Peptide, Scaffold-CSG-Peptide, Scaffold-CS-Peptide,Scaffold-KGGGG (SEQ ID NO:633)-Peptide, Scaffold-KGGG (SEQ IDNO:634)-Peptide, Scaffold-KGG-Peptide, Scaffold-KG-Peptide,Scaffold-K-Peptide.

In an embodiment, the peptide consists of any of the antigenic PCSK9peptide disclosed herein and the scaffold consists of any of theimmunogenic carrier disclosed herein, preferably a VLP.

Exemplary combinations of conjugations using various linkers and doublyconstrained peptides are provided below, where the carrier can be theidentical monomer of a carrier or a differential monomer of a carrier.(In the example below, the GC linker can be substituted by any of the GKlinker or GSC linker exemplified above or any other known to thoseskilled in the art):

Carrier-CGGGGG (SEQ ID NO:635)-Peptide-GGGGGC (SEQ ID NO:621)-carrier,Carrier-CGGGG (SEQ ID NO:636)-Peptide-GGGGC (SEQ ID NO:622)-carrier,Carrier-CGGGG (SEQ ID NO:6326-Peptide-GGGGC (SEQ ID NO:622)-carrier,Carrier-CGGG (SEQ ID NO:620)-Peptide-GGGC (SEQ ID NO:619)-carrier,Carrier-CG-Peptide-GC-carrier, Carrier-CG-Peptide-C-carrier,Carrier-C-Peptide-C-carrier.

In an embodiment, the peptide consists of any of the antigenic PCSK9peptide disclosed herein and the carrier consists of any of theimmunogenic carrier disclosed herein, preferably a VLP.

In an embodiment, the invention relates to an immunogen comprising anantigenic PCSK9 peptide consisting of, or consisting essentially of, anamino acid sequence selected from the group consisting of SEQ ID Nos: 1to 312, 330 to 398 and 420 to 588, wherein said antigenic antigenicPCSK9 peptide further comprises at its C-terminus or at its N-terminus acysteine which is chemically cross linked to an immunogenic carrier viaa thioether linkage. In a preferred embodiment, said immunogenic carrieris selected from the group consisting of DT (Diphtheria toxin), TT(tetanus toxid) or fragment C of TT, PD (Haemophilus influenzae proteinD), CRM197, other DT point mutants, such as CRM176, CRM228, CRM 45, CRM9, CRM102, CRM 103 and CRM107. Preferably said immunogenic carrier isCRM197.

In an embodiment, the invention relates to an immunogen comprising anantigenic PCSK9 peptide consisting of, or consisting essentially of, anamino acid sequence selected from the group consisting of 1 to 312, 330to 398 and 420 to 588, wherein said antigenic antigenic PCSK9 peptidefurther comprises at its C-terminus or at its N-terminus a cysteinewhich is chemically cross linked to an immunogenic carrier via athioether linkage using SMPH(Succinimidyl-6-[β-maleimidopropionamido]hexanoate) or BAANS(bromoacetic acid N-hydroxysuccinimide ester) as cross linker. In apreferred embodiment, said immunogenic carrier is selected from thegroup consisting of DT (Diphtheria toxin), TT (tetanus toxid) orfragment C of TT, PD (Haemophilus influenzae protein D, CRM197, other DTpoint mutants, such as CRM176, CRM228, CRM 45, CRM 9, CRM102, CRM 103and CRM107. Preferably said immunogenic carrier is CRM197. In anembodiment, the invention relates to an immunogen comprising anantigenic PCSK9 peptide consisting of, or consisting essentially of, anamino acid sequence selected from the group consisting of SEQ ID Nos: 1to 312, 330 to 398 and 420 to 588, wherein said antigenic antigenicPCSK9 peptide further comprises at its C-terminus a cysteine which ischemically cross linked to an immunogenic carrier via a thioetherlinkage using SMPH (Succinimidyl-6-[β-maleimidopropionamido]hexanoate)or BAANS (bromoacetic acid N-hydroxysuccinimide ester) as cross linker,said linkage being between a lysine residue of CRM197 and the cysteineresidue of said antigenic peptide.

Compositions Comprising an Antigenic PCSK9 Peptide of the Invention

The present invention further relates to compositions, particularlyimmunogenic compositions also referred to as “subject immunogeniccompositions”, comprising an antigenic PCSK9 peptide of the invention,preferably linked to an immunogenic carrier, and optionally at least oneadjuvant. Such immunogenic compositions, particularly when formulated aspharmaceutical compositions, are deemed useful to prevent, treat oralleviate PCSK9-related disorders.

In some embodiments, a subject immunogenic composition according to theinvention comprises an antigenic PCSK9 peptide, optionally comprising alinker, comprising an amino acid sequence selected from SEQ ID Nos 1 to328, 330 to 398, and 401 to 588 and functionally active variantsthereof. In some embodiment, said antigenic PCSK9 peptide is linked toan immunogenic carrier, preferably a DT, CRM197 or a VLP, morepreferably to a HBcAg, HBsAg, Qbeta, PP7, PPV or Norwalk Virus VLP.

In a preferred embodiment, a subject immunogenic composition accordingto the invention comprises an antigenic PCSK9 peptide, optionallycomprising a linker, comprising an amino acid sequence selected from SEQID Nos 1 to 328, 330 to 398 and 401 to 588, and functionally activevariants thereof linked to a VLP, preferably a Qbeta VLP.

In a preferred embodiment, a subject immunogenic composition accordingto the invention comprises an antigenic PCSK9 peptide optionallycomprising a linker, comprising an amino acid sequence selected from SEQID Nos 1 to 328, 330 to 398, and 401 to 588 and functionally activevariants thereof linked to CRM197.

A subject immunogenic composition comprising an antigenic PCSK9 peptideaccording to the invention can be formulated in a number of ways, asdescribed in more detail below.

In some embodiments, a subject immunogenic composition comprises singlespecies of antigenic PCSK9 peptide, e.g., the immunogenic compositioncomprises a population of antigenic PCSK9 peptides, substantially all ofwhich have the same amino acid sequence. In other embodiments, a subjectimmunogenic composition comprises two or more different antigenic PCSK9peptides, e.g., the immunogenic composition comprises a population ofantigenic PCSK9 peptides, the members of which population can differ inamino acid sequence. A subject immunogenic composition can comprise fromtwo to about 20 different antigenic PCSK9 peptides, e.g., a subjectimmunogenic composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15,or 15-20 different antigenic PCSK9 peptides, each having an amino acidsequence that differs from the amino acid sequences of the otherantigenic PCSK9 peptides.

In other embodiments, a subject immunogenic composition comprises amultimerized antigenic PCSK9 polypeptide, as described above. As usedherein, the terms “immunogenic composition comprising an antigenic PCSK9peptide” or “immunogenic composition of the invention” or “subjectimmunogenic composition” refers to an immunogenic composition comprisingeither single species (multimerized or not) or multiple species ofantigenic PCSK9 peptide(s) coupled or not to an immunogenic carrier.Where two or more peptides are used coupled to a carrier, the peptidemay be coupled to the same carrier molecule or individually coupled tocarrier molecules and then combined to produce an immunogeniccomposition.

Another aspect of the invention relates to methods for producing animmunogen according to the invention, said method comprising coupling anantigenic PCSK9 peptide to an immunogenic carrier. In one embodiment,said coupling is chemical.

Adjuvants

In some embodiments, a subject immunogenic composition comprises atleast one adjuvant. Suitable adjuvants include those suitable for use inmammals, preferably in humans. Examples of known suitable adjuvants thatcan be used in humans include, but are not necessarily limited to, alum,aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5%w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21(saponin adjuvant), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylatedMPL), extracts from Aquilla, ISCOMS (see, e.g., Sjölander et al. (1998)J. Leukocyte Biol. 64:713; WO90/03184, WO96/11711, WO 00/48630,WO98/36772, WO00/41720, WO06/134423 and WO07/026190), LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, TiterMaxclassic, TiterMax Gold, interleukins, and the like. For veterinaryapplications including but not limited to animal experimentation, onecan use Freund's adjuvant, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (WO90/14837; Chapter 10 in Vaccine design: the subunitand adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80 (polyoxyethylene sorbitanmono-oleate), and 0.5% Span 85 (sorbitan trioleate) (optionallycontaining muramyl tri-peptide covalently linked to dipalmitoylphosphatidylethanolamine (MTP-PE)) formulated into submicron particlesusing a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80,5% pluronic-blocked polymer L121, and thr-MDP either microfluidized intoa submicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components such as monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (DETOX™); (2) saponin adjuvants, such as QS21, STIMULON™(Cambridge Bioscience, Worcester, Mass.), Abisco® (Isconova, Sweden), orIscomatrix® (Commonwealth Serum Laboratories, Australia), may be used orparticles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g.WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g.gamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally inthe substantial absence of alum when used with pneumococcal saccharidese.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine2000, 19, 618-622; Krieg Curr opin Mol Ther2001 3:15-24; Roman et al.,Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94,10833-10837; Davis et al, J. Immunol, 1998, 160, 870-876; Chu et al., J.Exp. Med, 1997, 186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997,27, 2340-2344; Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg etal., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93,2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840-1845; Cowdery etal, J. Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996,167, 72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873;Stacey et al, J. Immunol., 1996, 157, 2116-2122; Messina et al, J.Immunol, 1991, 147, 1759-1764; Yi et al, J. Immunol, 1996, 157,4918-4925; Yi et al, J. Immunol, 1996, 157, 5394-5402; Yi et al, J.Immunol, 1998, 160, 4755-4761; and Yi et al, J. Immunol, 1998, 160,5898-5906; International patent applications WO96/02555, WO98/16247,WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e.containing at least one CG dinucleotide, where the cytosine isunmethylated; (8) a polyoxyethylene ether or a polyoxyethylene estere.g. WO99/52549; (9) a polyoxyethylene sorbitan ester surfactant incombination with an octoxynol (WO01/21207) or a polyoxyethylene alkylether or ester surfactant in combination with at least one additionalnon-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponinand an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)(WO00/62800); (11) an immunostimulant and a particle of metal salt e.g.WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241;(13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a sterol) e.g.WO98/57659; (14) other substances that act as immunostimulating agentsto enhance the efficacy of the composition, such as Muramyl peptidesinclude N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), (15) ligands for toll-like receptors (TLR), natural orsynthesized (e.g. as described in Kanzler et al 2007, Nature Medicine13, p 1552-9), including TLR3 ligands such as polyl:C and similarcompounds such as Hiltonol and Ampligen.

In a particular embodiment, said adjuvant is an immunostimulatoryoligonucleotide and more preferably a CpG oligonucleotide. A CpGoligonucleotide as used herein refers to an immunostimulatory CpGoligodeoxynucleotide (CpG ODN), and accordingly these terms are usedinterchangeably unless otherwise indicated. Immunostimulatory CpGoligodeoxynucleotides contain one or more immunostimulatory CpG motifsthat are unmethylated cytosine-guanine dinucleotides, optionally withincertain preferred base contexts. The methylation status of the CpGimmunostimulatory motif generally refers to the cytosine residue in thedinucleotide. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is an oligonucleotide which contains a5′ unmethylated cytosine linked by a phosphate bond to a 3′ guanine, andwhich activates the immune system through binding to Toll-like receptor9 (TLR-9). In another embodiment the immunostimulatory oligonucleotidemay contain one or more methylated CpG dinucleotides, which willactivate the immune system through TLR9 but not as strongly as if theCpG motif(s) was/were unmethylated. CpG immunostimulatoryoligonucleotides may comprise one or more palindromes that in turn mayencompass the CpG dinucleotide. CpG oligonucleotides have been describedin a number of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068.

Different classes of CpG immunostimulatory oligonucleotides have beenidentified. These are referred to as A, B, C and P class, and aredescribed in greater detail below. Methods of the invention embrace theuse of these different classes of CpG immunostimulatoryoligonucleotides.

Any of the classes may be subjugated to an E modification which enhancesits potency. An E modification may be a halogen substitution for the 5′terminal nucleotide; examples of such substitutions include but are notlimited to bromo-uridine or iodo-uridine substitutions. An Emodification can also include an ethyl-uridine substitution for the 5′terminal nucleotide.

The “A class” CpG immunostimulatory oligonucleotides are characterizedfunctionally by the ability to induce high levels of interferon-alpha(IFN-α) from plasmacytoid dendritic cells (pDC) and inducing NK cellactivation while having minimal effects on B cell activation.Structurally, this class typically has stabilized poly-G sequences at 5′and 3′ ends. It also has a palindromic phosphodiester CpGdinucleotide-containing sequence of at least 6 nucleotides, for examplebut not necessarily, it contains one of the following hexamerpalindromes: GACGTC, AGCGCT, or AACGTT described by Yamamoto andcolleagues. Yamamoto S et al. J. Immunol 148:4072-6 (1992). A class CpGimmunostimulatory oligonucleotides and exemplary sequences of this classhave been described in U.S. Non-Provisional patent application Ser. No.09/672,126 and published PCT application PCT/USOO/26527 (WO 01/22990),both filed on Sep. 27, 2000.

In an embodiment, the “A class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence: 5′ GGGGACGACGTCGTGGGGGGG 3′ (SEQ IDNO:637) Some non-limiting examples of A-Class oligonucleotides include:5′ G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3′ (SEQ ID NO:637);wherein * refers to a phosphorothioate bond and _ refers to aphosphodiester bond.

The B class CpG oligonucleotide sequences of the invention are thosebroadly described above as well as disclosed in published PCT PatentApplications PCT/US95/01570 and PCT/US97/19791, and in U.S. Pat. Nos.6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116 and 6,339,068.Exemplary sequences include but are not limited to those disclosed inthese latter applications and patents.

In an embodiment, the “B class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID No 589) 5′ TCGTCGTTTTTCGGTGCTTTT 3′,  or (SEQ ID No 590) 5′TCGTCGTTTTTCGGTCGTTTT 3′ or (SEQ ID No 591) 5′TCGTCGTTTTGTCGTTTTGTCGTT 3′ or (SEQ ID No 592) 5′TCGTCGTTTCGTCGTTTTGTCGTT 3′, or (SEQ ID No 593) 5′TCGTCGTTTTGTCGTTTTTTTCGA 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

Some non-limiting examples of B-Class oligonucleotides include:

(SEQ ID NO: 589) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′,  or(SEQ ID NO: 590) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′ or(SEQ ID NO: 591) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3′,  or (SEQ ID NO: 592) 5′T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*  T 3′, or(SEQ ID NO: 593) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G* A 3′.

wherein * refers to a phosphorothioate bond.

The “C class” of CpG immunostimulatory oligonucleotides is characterizedfunctionally by the ability to activate B cells and NK cells and induceIFN-α. Structurally, this class typically includes a region with one ormore B class-type immunostimulatory CpG motifs, and a GC-rich palindromeor near-palindrome region that allows the molecules to form secondary(e.g., stem-loop) or tertiary (e.g., dimer) type structures. Some ofthese oligonucleotides have both a traditional “stimulatory” CpGsequence and a “GC-rich” or “B-cell neutralizing” motif. Thesecombination motif oligonucleotides have immune stimulating effects thatfall somewhere between the effects associated with traditional B classCpG oligonucleotides (i.e., strong induction of B cell activation anddendritic cell (DC) activation), and the effects associated with A classCpG ODN (i.e., strong induction of IFN-α and NK cell activation butrelatively poor induction of B cell and DC activation). Krieg A M et al.(1995) Nature 374:546-9; Ballas Z K et al. (1996) J Immunol 157:1840-5;Yamamoto S et al. (1992) J Immunol 148:4072-6.

The C class of combination motif immune stimulatory oligonucleotides mayhave either completely stabilized, (e.g., all phosphorothioate),chimeric (phosphodiester central region), or semi-soft (e.g.,phosphodiester within CpG motif) backbones. This class has beendescribed in U.S. patent application U.S. Ser. No. 10/224,523 filed onAug. 19, 2002. One stimulatory domain or motif of the C class CpGoligonucleotide is defined by the formula: 5′ X₁DCGHX₂ 3′. D is anucleotide other than C. C is cytosine. G is guanine. H is a nucleotideother than G. X₁ and X₂ are any nucleic acid sequence 0 to 10nucleotides long. X₁ may include a CG, in which case there is preferablya T immediately preceding this CG. In some embodiments, DCG is TCG. X₁is preferably from 0 to 6 nucleotides in length. In some embodiments, X₂does not contain any poly G or poly A motifs. In other embodiments, theimmunostimulatory oligonucleotide has a poly-T sequence at the 5′ end orat the 3′ end. As used herein, “poly-A” or “poly-T” shall refer to astretch of four or more consecutive A's or T's respectively, e.g., 5′AAAA 3′ or 5′ TTTT 3′. As used herein, “poly-G end” shall refer to astretch of four or more consecutive G's, e.g., 5′ GGGG 3′, occurring atthe 5′ end or the 3′ end of a nucleic acid. As used herein, “poly-Goligonucleotide” shall refer to an oligonucleotide having the formula 5′X₁X₂GGGX₃X₄ 3′ wherein X₁, X₂, X₃, and X₄ are nucleotides and preferablyat least one of X₃ and X₄ is a G. Some preferred designs for the B cellstimulatory domain under this formula comprise TTTTTCG, TCG, TTCG,TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT, TCGTCGT.

The second motif of the C class CpG oligonucleotide is referred to aseither P or N and is positioned immediately 5′ to X₁ or immediately 3′to X₂.

N is a B cell neutralizing sequence that begins with a CGG trinucleotideand is at least 10 nucleotides long. A B cell neutralizing motifincludes at least one CpG sequence in which the CG is preceded by a C orfollowed by a G (Krieg A M et al. (1998) Proc Natl Acad Sd USA95:12631-12636) or is a CG containing DNA sequence in which the C of theCG is methylated. Neutralizing motifs or sequences have some degree ofimmunostimulatory capability when present in an otherwisenon-stimulatory motif, but when present in the context of otherimmunostimulatory motifs serve to reduce the immunostimulatory potentialof the other motifs.

P is a GC-rich palindrome containing sequence at least 10 nucleotideslong.

As used herein, “palindrome” and equivalently “palindromic sequence”shall refer to an inverted repeat, i.e., a sequence such asABCDEE′D′C′B′A′ in which A and A′, B and B′, etc., are bases capable offorming the usual Watson-Crick base pairs.

As used herein, “GC-rich palindrome” shall refer to a palindrome havinga base composition of at least two-thirds G's and Cs. In someembodiments the GC-rich domain is preferably 3′ to the “B cellstimulatory domain”. In the case of a 10-base long GC-rich palindrome,the palindrome thus contains at least 8 G's and Cs. In the case of a12-base long GC-rich palindrome, the palindrome also contains at least 8G's and Cs. In the case of a 14-mer GC-rich palindrome, at least tenbases of the palindrome are G's and Cs. In some embodiments the GC-richpalindrome is made up exclusively of G's and Cs.

In some embodiments the GC-rich palindrome has a base composition of atleast 81% G's and Cs. In the case of such a 10-base long GC-richpalindrome, the palindrome thus is made exclusively of G's and Cs. Inthe case of such a 12-base long GC-rich palindrome, it is preferred thatat least ten bases (83%) of the palindrome are G's and Cs. In somepreferred embodiments, a 12-base long GC-rich palindrome is madeexclusively of G's and Cs. In the case of a 14-mer GC-rich palindrome,at least twelve bases (86%) of the palindrome are G's and Cs. In somepreferred embodiments, a 14-base long GC-rich palindrome is madeexclusively of G's and Cs. The Cs of a GC-rich palindrome can beunmethylated or they can be methylated.

In general this domain has at least 3 Cs and Gs, more preferably 4 ofeach, and most preferably 5 or more of each. The number of Cs and Gs inthis domain need not be identical. It is preferred that the Cs and Gsare arranged so that they are able to form a self-complementary duplex,or palindrome, such as CCGCGCGG. This may be interrupted by As or Ts,but it is preferred that the self-complementarity is at least partiallypreserved as for example in the motifs CGACGTTCGTCG (SEQ ID NO:639) orCGGCGCCGTGCCG (SEQ ID NO:640). When complementarity is not preserved, itis preferred that the non-complementary base pairs be TG. In a preferredembodiment there are no more than 3 consecutive bases that are not partof the palindrome, preferably no more than 2, and most preferablyonly 1. In some embodiments, the GC-rich palindrome includes at leastone CGG trimer, at least one CCG trimer, or at least one CGCG tetramer.In other embodiments, the GC-rich palindrome is not CCCCCCGGGGGG (SEQ IDNO:641) or GGGGGGCCCCCC (SEQ ID NO:642), CCCCCGGGGG (SEQ ID NO:643) orGGGGGCCCCC (SEQ ID NO:644).

At least one of the G's of the GC rich region may be substituted with aninosine (I). In some embodiments, P includes more than one I.

In certain embodiments, the immunostimulatory oligonucleotide has one ofthe following formulas 5′ NX₁DCGHX₂ 3′, 5′ X₁DCGHX₂N 3′, 5′ PX₁DCGHX₂3′, 5′ X₁DCGHX₂P 3′, 5′ X₁DCGHX₂PX₃ 3′, 5′ X₁DCGHPX₃ 3′, 5′ DCGHX₂PX₃3′, 5′ TCGHX₂PX3 3′, 5′ DCGHPX₃ 3′ or 5′DCGHP 3′.

The invention provides other immune stimulatory oligonucleotides definedby a formula 5′ N₁PyGN₂P 3′. N₁ is any sequence 1 to 6 nucleotides long.Py is a pyrimidine. G is guanine. N₂ is any sequence 0 to 30 nucleotideslong. P is a GC-rich palindrome containing a sequence at least 10nucleotides long.

N₁ and N₂ may contain more than 50% pyrimidines, and more preferablymore than 50% T. N₁ may include a CG, in which case there is preferablya T immediately preceding this CG. In some embodiments, N1PyG is TCG,and most preferably a TCGN₂, where N₂ is not G.

N₁PyGN₂P may include one or more inosine (I) nucleotides. Either the Cor the G in N₁ may be replaced by inosine, but the Cpl is preferred tothe IpG. For inosine substitutions such as IpG, the optimal activity maybe achieved with the use of a “semi-soft” or chimeric backbone, wherethe linkage between the IG or the Cl is phosphodiester. N1 may includeat least one Cl, TCl, IG or TIG motif.

In certain embodiments N₁PyGN₂ is a sequence selected from the groupconsisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT,and TCGTCGT.

In an embodiment, the “C class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID No 594) 5′ TCGCGTCGTTCGGCGCGCGCCG 3′,  or (SEQ ID No 595) 5′TCGTCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID No 596) 5′TCGGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID No 597) 5′TCGGACGTTCGGCGCGCCG 3′,  or (SEQ ID No 598) 5′ TCGCGTCGTTCGGCGCGCCG 3′, or (SEQ ID No 599) 5′ TCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID No 600) 5′TCGACGTTCGGCGCGCCG 3′,  or (SEQ ID No 601) 5′ TCGCGTCGTTCGGCGCCG 3′,  or(SEQ ID No 602) 5′ TCGCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID No 603) 5′TCGTCGTTTTCGGCGCGCGCCG 3′,  or (SEQ ID No 604) 5′TCGTCGTTTTCGGCGGCCGCCG 3′,  or (SEQ ID No 605) 5′TCGTCGTTTTACGGCGCCGTGCCG 3′,  or (SEQ ID No 606) 5′TCGTCGTTTTCGGCGCGCGCCGT 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide.

Some non-limiting examples of C-Class oligonucleotides include:

(SEQ ID NO: 594)  5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 595) 5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C* G 3′,or (SEQ ID NO: 596) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 597) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 598) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 599) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 600) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 601) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3′, or(SEQ ID NO: 602) 5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 603) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 604) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3′, or(SEQ ID NO: 605) 5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C* G 3′,or (SEQ ID NO: 606) 5′ T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G* T 3′

wherein * refers to a phosphorothioate bond and _ refers to aphosphodiester bond.

In any of these sequences, an ethyl-uridine or a halogen may substitutefor the 5′ T; examples of halogen substitutions include but are notlimited to bromo-uridine or iodo-uridine substitutions.

The “P class” CpG immunostimulatory oligonucleotides have been describedin WO2007/095316 and are characterized by the fact that they containduplex forming regions such as, for example, perfect or imperfectpalindromes at or near both the 5′ and 3′ ends, giving them thepotential to form higher ordered structures such as concatamers. Theseoligonucleotides referred to as P-Class oligonucleotides have theability in some instances to induce much high levels of IFN-α secretionthan the C-Class. The P-Class oligonucleotides have the ability tospontaneously self-assemble into concatamers either in vitro and/or invivo. Without being bound by any particular theory for the method ofaction of these molecules, one potential hypothesis is that thisproperty endows the P-Class oligonucleotides with the ability to morehighly crosslink TLR9 inside certain immune cells, inducing a distinctpattern of immune activation compared to the previously describedclasses of CpG oligonucleotides.

In an embodiment, the CpG oligonucleotide for use in the presentinvention is a P class CpG oligonucleotide containing a 5′ TLRactivation domain and at least two palindromic regions, one palindromicregion being a 5′ palindromic region of at least 6 nucleotides in lengthand connected to a 3′ palindromic region of at least 8 nucleotides inlength either directly or through a spacer, wherein the oligonucleotideincludes at least one YpR dinucleotide. In an embodiment, saidoligonucleotide is not T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G(SEQ ID NO:595). In one embodiment the P class CpG oligonucleotideincludes at least one unmethylated CpG dinucleotide. In anotherembodiment the TLR activation domain is TCG, TTCG, TTTCG, TYpR, TTYpR,TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. In yet another embodiment theTLR activation domain is within the 5′ palindromic region. In anotherembodiment the TLR activation domain is immediately 5′ to the 5′palindromic region. In still another embodiment the 5′ palindromicregion is at least 8 nucleotides in length. In another embodiment the 3′palindromic region is at least 10 nucleotides in length. In anotherembodiment the 5′ palindromic region is at least 10 nucleotides inlength. In yet another embodiment the 3′ palindromic region includes anunmethylated CpG dinucleotide. In another embodiment the 3′ palindromicregion includes two unmethylated CpG dinucleotides. In anotherembodiment the 5′ palindromic region includes an unmethylated CpGdinucleotide. In yet another embodiment the 5′ palindromic regionincludes two unmethylated CpG dinucleotides. In another embodiment the5′ and 3′ palindromic regions have a duplex stability value of at least25. In another embodiment the 5′ and 3′ palindromic regions have aduplex stability value of at least 30. In another embodiment the 5′ and3′ palindromic regions have a duplex stability value of at least 35. Inanother embodiment the 5′ and 3′ palindromic regions have a duplexstability value of at least 40. In another embodiment the 5′ and 3′palindromic regions have a duplex stability value of at least 45. Inanother embodiment the 5′ and 3′ palindromic regions have a duplexstability value of at least 50. In another embodiment the 5′ and 3′palindromic regions have a duplex stability value of at least 55. Inanother embodiment the 5′ and 3′ palindromic regions have a duplexstability value of at least 60. In another embodiment the 5′ and 3′palindromic regions have a duplex stability value of at least 65.

In one embodiment the two palindromic regions are connected directly. Inanother embodiment the two palindromic regions are connected via a 3‘-3’ linkage. In another embodiment the two palindromic regions overlapby one nucleotide. In yet another embodiment the two palindromic regionsoverlap by two nucleotides. In another embodiment the two palindromicregions do not overlap. In another embodiment the two palindromicregions are connected by a spacer. In one embodiment the spacer is anucleic acid having a length of 1-50 nucleotides. In another embodimentthe spacer is a nucleic acid having a length of 1 nucleotide. In anotherembodiment the spacer is a non-nucleotide spacer. In one embodiment thenon-nucleotide spacer is a D-spacer. In another embodiment thenon-nucleotide spacer is a linker. In one embodiment the oligonucleotidehas the formula 5′ XP₁SP₂T 3′, wherein X is the TLR activation domain,P₁ is a palindrome, S is a spacer, P₂ is a palindrome, and T is a 3′tail of 0-100 nucleotides in length. In one embodiment X is TCG, TTCG,or TTTCG. In another embodiment T is 5-50 nucleotides in length. In yetanother embodiment T is 5-10 nucleotides in length. In one embodiment Sis a nucleic acid having a length of 1-50 nucleotides. In anotherembodiment S is a nucleic acid having a length of 1 nucleotide. Inanother embodiment S is a non-nucleotide spacer. In one embodiment thenon-nucleotide spacer is a D-spacer. In another embodiment thenon-nucleotide spacer is a linker. In another embodiment theoligonucleotide is not an antisense oligonucleotide or a ribozyme. Inone embodiment P₁ is A and T rich. In another embodiment P₁ includes atleast 4 Ts. In another embodiment P₂ is a perfect palindrome. In anotherembodiment P2 is G-C rich. In still another embodiment P₂ isCGGCGCX₁GCGCCG, where X₁ is T or nothing.

In one embodiment the oligonucleotide includes at least onephosphorothioate linkage. In another embodiment all internucleotidelinkages of the oligonucleotide are phosphorothioate linkages. Inanother embodiment the oligonucleotide includes at least onephosphodiester-like linkage. In another embodiment thephosphodiester-like linkage is a phosphodiester linkage. In anotherembodiment a lipophilic group is conjugated to the oligonucleotide. Inone embodiment the lipophilic group is cholesterol.

In an embodiment, the TLR-9 agonist for use in the present invention isa P class CpG oligonucleotide with a 5′ TLR activation domain and atleast two complementarity-containing regions, a 5′ and a 3′complementarity-containing region, each complementarity-containingregion being at least 8 nucleotides in length and connected to oneanother either directly or through a spacer, wherein the oligonucleotideincludes at least one pyrimidine-purine (YpR) dinucleotide, and whereinat least one of the complementarity-containing regions is not a perfectpalindrome. In one embodiment the oligonucleotide includes at least oneunmethylated CpG dinucleotide. In another embodiment the TLR activationdomain is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT,or TTTT. In another embodiment the TLR activation domain is within the5′ complementarity-containing region. In another embodiment the TLRactivation domain is immediately 5′ to the 5′ complementarity-containingregion. In another embodiment the 3′ complementarity-containing regionis at least 10 nucleotides in length. In yet another embodiment the 5′complementarity-containing region is at least 10 nucleotides in length.In one embodiment the 3′ complementarity-containing region includes anunmethylated CpG dinucleotide. In another embodiment the 3′complementarity-containing region includes two unmethylated CpGdinucleotides. In yet another embodiment the 5′complementarity-containing region includes an unmethylated CpGdinucleotide. In another embodiment the 5′ complementarity-containingregion includes two unmethylated CpG dinucleotides. In anotherembodiment the complementarity-containing regions include at least onenucleotide analog. In another embodiment the complementarity-containingregions form an intramolecular duplex. In one embodiment theintramolecular duplex includes at least one non-Watson Crick base pair.In another embodiment the non-Watson Crick base pair is G-T, G-A, G-G,or C-A. In one embodiment the complementarity-containing regions formintermolecular duplexes. In another embodiment at least one of theintermolecular duplexes includes at least one non-Watson Crick basepair. In another embodiment the non-Watson Crick base pair is G-T, G-A,G-G, or C-A. In yet another embodiment the complementarity-containingregions contain a mismatch. In still another embodiment thecomplementarity-containing regions contain two mismatches. In anotherembodiment the complementarity-containing regions contain an interveningnucleotide. In another embodiment the complementarity-containing regionscontain two intervening nucleotides.

In one embodiment the 5′ and 3′ complementarity-containing regions havea duplex stability value of at least 25. In another embodiment the 5′and 3′ complementarity-containing regions have a duplex stability valueof at least 30. In another embodiment the 5′ and 3′complementarity-containing regions have a duplex stability value of atleast 35. In another embodiment the complementarity-containing regionshave a duplex stability value of at least 40. In another embodiment thecomplementarity-containing regions have a duplex stability value of atleast 45. In another embodiment the complementarity-containing regionshave a duplex stability value of at least 50. In another embodiment thecomplementarity-containing regions have a duplex stability value of atleast 55. In another embodiment the complementarity-containing regionshave a duplex stability value of at least 60. In another embodiment thecomplementarity-containing regions have a duplex stability value of atleast 65.

In another embodiment the two complementarity-containing regions areconnected directly. In another embodiment the two palindromic regionsare connected via a 3′-3′ linkage. In yet another embodiment the twocomplementarity-containing regions overlap by one nucleotide. In anotherembodiment the two complementarity-containing regions overlap by twonucleotides. In another embodiment the two complementarity-containingregions do not overlap. In another embodiment the twocomplementarity-containing regions are connected by a spacer. In anotherembodiment the spacer is a nucleic acid having a length of 1-50nucleotides. In another embodiment the spacer is a nucleic acid having alength of 1 nucleotide. In one embodiment the spacer is a non-nucleotidespacer. In another embodiment the non-nucleotide spacer is a D-spacer.In yet another embodiment the non-nucleotide spacer is a linker.

In one embodiment the P-class oligonucleotide has the formula 5′ XNSPT3′, wherein X is the TLR activation domain, N is a non-perfectpalindrome, P is a palindrome, S is a spacer, and T is a 3′ tail of0-100 nucleotides in length. In another embodiment X is TCG, TTCG, orTTTCG. In another embodiment T is 5-50 nucleotides in length. In anotherembodiment T is 5-10 nucleotides in length. In another embodiment S is anucleic acid having a length of 1-50 nucleotides. In another embodimentS is a nucleic acid having a length of 1 nucleotide. In anotherembodiment S is a non-nucleotide spacer. In another embodiment thenon-nucleotide spacer is a D-spacer. In another embodiment thenon-nucleotide spacer is a linker. In another embodiment theoligonucleotide is not an antisense oligonucleotide or a ribozyme. Inanother embodiment N is A and T rich. In another embodiment N isincludes at least 4 Ts. In another embodiment P is a perfect palindrome.In another embodiment P is G-C rich. In another embodiment P isCGGCGCX₁GCGCCG, wherein X₁ is T or nothing. In another embodiment theoligonucleotide includes at least one phosphorothioate linkage. Inanother embodiment all interaucleotide linkages of the oligonucleotideare phosphorothioate linkages. In another embodiment the oligonucleotideincludes at least one phosphodiester-like linkage. In another embodimentthe phosphodiester-like linkage is a phosphodiester linkage. In anotherembodiment a lipophilic group is conjugated to the oligonucleotide. Inone embodiment the lipophilic group is cholesterol.

In an embodiment, the “P class” CpG oligonucleotides of the inventionhas the following nucleic acid sequence: 5′ TCGTCGACGATCGGCGCGCGCCG 3′(SEQ ID No 607).

In said sequences, all of the linkages may be all phosphorothioatebonds. In another embodiment, one or more of the linkages may bephosphodiester, preferably between the “C” and the “G” of the CpG motifmaking a semi-soft CpG oligonucleotide. In any of these sequences, anethyl-uridine or a halogen may substitute for the 5′ T; examples ofhalogen substitutions include but are not limited to bromo-uridine oriodo-uridine substitutions.

A non-limiting example of P-Class oligonucleotides include:

(SEQ ID NO: 607) 5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3′

wherein * refers to a phosphorothioate bond and _ refers to aphosphodiester bond.

In an embodiment, all the internucleotide linkage of the CpGoligonucleotides disclosed herein are phosphodiester bonds (“soft”oligonucleotides, as described in the PCT application WO2007/026190). Inanother embodiment, CpG oligonucleotides of the invention are renderedresistant to degradation (e.g., are stabilized). A “stabilizedoligonucleotide” refers to an oligonucleotide that is relativelyresistant to in vivo degradation (e.g. via an exo- or endo-nuclease).Nucleic acid stabilization can be accomplished via backbonemodifications. Oligonucleotides having phosphorothioate linkages providemaximal activity and protect the oligonucleotide from degradation byintracellular exo- and endo-nucleases.

The immunostimulatory oligonucleotides may have a chimeric backbone,which have combinations of phosphodiester and phosphorothioate linkages.For purposes of the instant invention, a chimeric backbone refers to apartially stabilized backbone, wherein at least one internucleotidelinkage is phosphodiester or phosphodiester-like, and wherein at leastone other internucleotide linkage is a stabilized internucleotidelinkage, wherein the at least one phosphodiester or phosphodiester-likelinkage and the at least one stabilized linkage are different. When thephosphodiester linkage is preferentially located within the CpG motifsuch molecules are called “semi-soft” as described in the PCTapplication WO2007/026190.

Other modified oligonucleotides include combinations of phosphodiester,phosphorothioate, methylphosphonate, methylphosphorothioate,phosphorodithioate, and/or p-ethoxy linkages.

Since boranophosphonate linkages have been reported to be stabilizedrelative to phosphodiester linkages, for purposes of the chimeric natureof the backbone, boranophosphonate linkages can be classified either asphosphodiester-like or as stabilized, depending on the context. Forexample, a chimeric backbone according to the instant invention could,in some embodiments, includes at least one phosphodiester(phosphodiester or phosphodiester-like) linkage and at least oneboranophosphonate (stabilized) linkage. In other embodiments, a chimericbackbone according to the instant invention could includeboranophosphonate (phosphodiester or phosphodiester-like) andphosphorothioate (stabilized) linkages. A “stabilized internucleotidelinkage” shall mean an internucleotide linkage that is relativelyresistant to in vivo degradation (e.g., via an exo- or endo-nuclease),compared to a phosphodiester internucleotide linkage. Preferredstabilized internucleotide linkages include, without limitation,phosphorothioate, phosphorodithioate, methylphosphonate, andmethylphosphorothioate. Other stabilized internucleotide linkagesinclude, without limitation, peptide, alkyl, dephospho, and others asdescribed above.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al. (1990) Chem Rev 90:544; GoodchildJ (1990) Bioconjugate Chem 1:165. Methods for preparing chimericoligonucleotides are also known. For instance patents issued to Uhlmannet al have described such techniques.

Mixed backbone modified ODN may be synthesized as described in the PCTapplication WO2007/026190.

The oligonucleotides of the invention can also include othermodifications. These include nonionic DNA analogs, such as alkyl- andaryl-phosphates (in which the charged phosphonate oxygen is replaced byan alkyl or aryl group), phosphodiester and alkylphosphotriesters, inwhich the charged oxygen moiety is alkylated. Nucleic acids whichcontain diol, such as tetraethyleneglycol or hexaethyleneglycol, ateither or both termini have also been shown to be substantiallyresistant to nuclease degradation.

The size of the CpG oligonucleotide (i.e., the number of nucleotideresidues along the length of the oligonucleotide) also may contribute tothe stimulatory activity of the oligonucleotide. For facilitating uptakeinto cells, CpG oligonucleotide of the invention preferably have aminimum length of 6 nucleotide residues. Oligonucleotides of any sizegreater than 6 nucleotides (even many kb long) are capable of inducingan immune response if sufficient immunostimulatory motifs are present,because larger oligonucleotides are degraded inside cells. In certainembodiments, the CpG oligonucleotides are 6 to 100 nucleotides long,preferentially 8 to 30 nucleotides long. In important embodiments,nucleic acids and oligonucleotides of the invention are not plasmids orexpression vectors.

In an embodiment, the CpG oligonucleotide disclosed herein comprisesubstitutions or modifications, such as in the bases and/or sugars asdescribed at paragraph 134 to 147 of WO2007/026190.

In an embodiment, the CpG oligonucleotide of the present invention ischemically modified. Examples of chemical modifications are known to theskilled person and are described, for example in Uhlmann E. et al.(1990), Chem. Rev. 90:543, S. Agrawal, Ed., Humana Press, Totowa, USA1993; Crooke, S. T. et al. (1996) Annu. Rev. Pharmacol. Toxicol.36:107-129; and Hunziker J. et al., (1995), Mod. Synth. Methods7:331-417. An oligonucleotide according to the invention may have one ormore modifications, wherein each modification is located at a particularphosphodiester internucleoside bridge and/or at a particular β-D-riboseunit and/or at a particular natural nucleoside base position incomparison to an oligonucleotide of the same sequence which is composedof natural DNA or RNA.

In some embodiments of the invention, CpG-containing nucleic acids mightbe simply mixed with immunogenic carriers according to methods known tothose skilled in the art (see, e.g. WO03/024480).

In a particular embodiment of the present invention, any of the vaccinedisclosed herein comprises from 20 μg to 20 mg of CpG oligonucleotide,preferably from 0.1 mg to 10 mg CpG oligonucleotide, preferably from 0.2mg to 5 mg CpG oligonucleotide, preferably from 0.3 mg to 3 mg CpGoligonucleotide, even preferably from 0.4 to 2 mg CpG oligonucleotide,even preferably from 0.5 to 1.5 mg CpG oligonucleotide. In a preferredembodiment, any of the vaccine disclosed herein comprises approximately0.5 to 1 mg CpG oligonucleotide.

Preferred adjuvants for use in the present invention are alum, QS21, CpGODN, alum in combination with CpG ODN, Iscomatrix and Iscomatrix incombination with CpG ODN.

PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

The invention also provides pharmaceutical compositions comprising anantigenic PCSK9 peptide of the invention or an immunogenic compositionthereof, in a formulation in association with one or morepharmaceutically acceptable excipient(s) and optionally combined withone or more adjuvants (as adjuvant described above). The term‘excipient’ is used herein to describe any ingredient other than theactive ingredient, i.e. the antigenic PCSK9 peptide of the inventioneventually coupled to an immunogenic carrier and optionally combinedwith one or more adjuvants. The choice of excipient(s) will to a largeextent depend on factors such as the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Some examplesof pharmaceutically acceptable excipients are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the active ingredient.

Pharmaceutical compositions of the present invention and methods fortheir preparation will be readily apparent to those skilled in the art.Such compositions and methods for their preparation may be found, forexample, in Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company, 1995). Pharmaceutical compositions are preferablymanufactured under GMP conditions.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

Any method for administering peptides, or proteins accepted in the artmay suitably be employed for the peptides or proteins of the invention.

The pharmaceutical compositions of the invention are typically suitablefor parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intracranial, intrasynovial injection or infusions; and kidney dialyticinfusion techniques. Preferred embodiments include the intravenous,subcutaneous, intradermal and intramuscular routes, even more preferredembodiments are the intramuscular or the subcutaneous routes.

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or inmulti-dose containers containing a preservative. Formulations forparenteral administration include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, and the like.Such formulations may further comprise one or more additionalingredients including, but not limited to, suspending, stabilizing, ordispersing agents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, microparticles, or in a liposomal preparation. Formulations forparenteral administration may be formulated to be immediate and/ormodified release. Modified release formulations include delayed-,sustained-, pulsed-, controlled-, targeted and programmed release.

For example, in one aspect, sterile injectable solutions can be preparedby incorporating the anti-PCSK9 peptide, preferably coupled to animmunogenic carrier, optionally in combination with one or moreadjuvants, in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

An exemplary, non-limiting pharmaceutical composition of the inventionis a formulation as a sterile aqueous solution having a pH that rangesfrom about 5.0 to about 6.5 and comprising from about 0.1 mg/mL to about20 mg/mL of a peptide of the invention, from about 1 millimolar to about100 millimolar of histidine buffer, from about 0.01 mg/mL to about 10mg/mL of polysorbate 80, from about 100 millimolar to about 400millimolar of trehalose, and from about 0.01 millimolar to about 1.0millimolar of disodium EDTA dihydrate.

The antigenic PCSK9 peptides of the invention can also be administeredintranasally or by inhalation, typically in the form of a dry powder(either alone, as a mixture, or as a mixed component particle, forexample, mixed with a suitable pharmaceutically acceptable excipient)from a dry powder inhaler, as an aerosol spray from a pressurisedcontainer, pump, spray, atomiser (preferably an atomiser usingelectrohydrodynamics to produce a fine mist), or nebuliser, with orwithout the use of a suitable propellant, or as nasal drops.

The pressurised container, pump, spray, atomizer, or nebuliser generallycontains a solution or suspension of an antibody of the inventioncomprising, for example, a suitable agent for dispersing, solubilising,or extending release of the active, a propellant(s) as solvent.

Prior to use in a dry powder or suspension formulation, the drug productis generally micronised to a size suitable for delivery by inhalation(typically less than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenisation, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the compound of theinvention, a suitable powder base and a performance modifier.

A suitable solution formulation for use in an atomiser usingelectrohydrodynamics to produce a fine mist may contain a suitable doseof the antigenic PCSK9 peptide of the invention per actuation and theactuation volume may for example vary from 1 μL to 100 μL.

Suitable flavours, such as menthol and levomenthol, or sweeteners, suchas saccharin or saccharin sodium, may be added to those formulations ofthe invention intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled-, targeted andprogrammed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve which delivers a metered amount. Units inaccordance with the invention are typically arranged to administer ametered dose or “puff” of an antibody of the invention. The overalldaily dose will typically be administered in a single dose or, moreusually, as divided doses throughout the day.

A pharmaceutical composition comprising an antigenic PCSK9 peptide mayalso be formulated for an oral route administration. Oral administrationmay involve swallowing, so that the compound enters the gastrointestinaltract, and/or buccal, lingual, or sublingual administration by which thecompound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include solid, semi-solidand liquid systems such as tablets; soft or hard capsules containingmulti- or nano-particulates, liquids, or powders; lozenges (includingliquid-filled); chews; gels; fast dispersing dosage forms; films;ovules; sprays; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsules(made, for example, from gelatin or hydroxypropylmethylcellulose) andtypically comprise a carrier, for example, water, ethanol, polyethyleneglycol, propylene glycol, methylcellulose, or a suitable oil, and one ormore emulsifying agents and/or suspending agents. Liquid formulationsmay also be prepared by the reconstitution of a solid, for example, froma sachet.

The compositions of the invention can be used to treat, alleviate orprevent PCSK9-mediated disorders or symptoms in a subject at risk orsuffering from such disorder or symptom by stimulating an immuneresponse in said subject by immunotherapy. Immunotherapy can comprise aninitial immunization followed by additional, e. g. one, two, three, ormore boosters.

An “immunologically effective amount” of an antigenic PCSK9 peptide ofthe invention, or composition thereof, is an amount that is delivered toa mammalian subject, either in a single dose or as part of a series,which is effective for inducing an immune response against PCSK9 in saidsubject. This amount varies depending upon the health and physicalcondition of the individual to be treated, the taxonomic group ofindividual to be treated, the capacity of the individual's immune systemto synthesize antibodies, the formulation of the vaccine, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.

A “pharmaceutically effective dose” or “therapeutically effective dose”is that dose required to treat or prevent, or alleviate one or morePCSK9-related disorder or symptom in a subject. The pharmaceuticallyeffective dose depends on inter alia the specific compound toadminister, the severity of the symptoms, the susceptibility of thesubject to side effects, the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration such ashealth and physical condition, concurrent medication, the capacity ofthe individual's immune system to synthesize antibodies, the degree ofprotection desired, and other factors that those skilled in the medicalarts will recognize. For prophylaxis purposes, the amount of peptide ineach dose is selected as an amount which induces an immunoprotectiveresponse without significant adverse side effects in typical vaccines.Following an initial vaccination, subjects may receive one or severalbooster immunisations adequately spaced.

It is understood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For example, antigenic PCSK9 peptides or pharmaceutical composition ofthe invention can be administered to a subject at a dose of about 0.1 μgto about 5 mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg toabout 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500μg, from about 500 μg to about 1 mg, from about 1 mg to about 2 mg, withoptional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4weeks, two months, three months, 6 months and/or a year later.

In some embodiments, a single dose of an antigenic PCSK9 peptide orpharmaceutical composition according to the invention is administered.In other embodiments, multiple doses of an antigenic PCSK9 peptide orpharmaceutical composition according to the invention are administered.The frequency of administration can vary depending on any of a varietyof factors, e.g., severity of the symptoms, degree of immunoprotectiondesired, whether the composition is used for prophylactic or curativepurposes, etc. For example, in some embodiments, an antigenic PCSK9peptide or pharmaceutical composition according to the invention isadministered once per month, twice per month, three times per month,every other week (qow), once per week (qw), twice per week (biw), threetimes per week (tiw), four times per week, five times per week, sixtimes per week, every other day (qod), daily (qd), twice a day (qid), orthree times a day (tid). When the composition of the invention is usedfor prophylaxis purposes, they will be generally administered for bothpriming and boosting doses. It is expected that the boosting doses willbe adequately spaced, or preferably given yearly or at such times wherethe levels of circulating antibody fall below a desired level. Boostingdoses may consist of the antigenic PCSK9 peptide in the absence of theoriginal immunogenic carrier molecule. Such booster constructs maycomprise an alternative immunogenic carrier or may be in the absence ofany carrier. Such booster compositions may be formulated either with orwithout adjuvant.

The duration of administration of an antigenic PCSK9 peptide accordingto the invention, e.g., the period of time over which an antigenic PCSK9peptide is administered, can vary, depending on any of a variety offactors, e.g., patient response, etc. For example, an antigenic PCSK9peptide can be administered over a period of time ranging from about oneday to about one week, from about two weeks to about four weeks, fromabout one month to about two months, from about two months to about fourmonths, from about four months to about six months, from about sixmonths to about eight months, from about eight months to about 1 year,from about 1 year to about 2 years, or from about 2 years to about 4years, or more.

A variety of treatment methods are also contemplated by the presentdisclosure, which methods comprise administering an antigenic PCSK9peptide according to the invention. Subject treatment methods includemethods of inducing an immune response in an individual to self-PCSK9,and methods of preventing, alleviating or treating a PCSK9-relateddisorder or symptom in an individual.

In one aspect, the present invention provides a method for treating,preventing or alleviating a PCSK9-related disorder or symptom in asubject, comprising administering a therapeutically effective amount ofan antigenic PCSK9 peptide of the invention, or immunogenic orpharmaceutical composition thereof, to said subject.

In another aspect, the present invention provides a method for inducingan immune response against self-PCSK9 in a subject, comprisingadministering a therapeutically or immunogenically effective amount ofan antigenic PCSK9 peptide of the invention, or immunogenic orpharmaceutical composition thereof, to said subject.

A PCSK9 related disease or a PCSK9 mediated disease is, for example, adisease where the inhibition of PCSK9 activity or the inhibition of theinteraction of PCSK9 with the LDL receptor could be beneficial.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a biological disorder and/or at least one of its attendantsymptoms. As used herein, to “alleviate” a disease, disorder orcondition means reducing the severity and/or occurrence frequency of thesymptoms of the disease, disorder, or condition. Further, referencesherein to “treatment” include references to curative, palliative andprophylactic treatment. Said subject is preferably human, and may beeither male or female, of any age.

Other aspects of the invention relate to an antigenic PCSK9 peptideaccording to the invention or of an immunogenic composition or apharmaceutical composition thereof, for use as a medicament, preferablyin treatment, alleviation or prophylaxis of PCSK9-related disorders.

In yet another aspect, the present invention provides the use of anantigenic PCSK9 peptide of the invention or of an immunogeniccomposition or a pharmaceutical composition thereof, in the manufactureof a medicament, preferably for treating a PCSK9-related disorder.

In particular, the invention relates to an antigenic PCSK9 peptide ofthe invention, or an immunogenic or pharmaceutical composition thereof,for use as a medicament preferably in treatment, alleviation orprophylaxis of diseases associated with an elevated level ofcholesterol.

In yet another aspect, the present invention provides the use of anantigenic PCSK9 peptide of the invention or of an immunogeniccomposition or a pharmaceutical composition thereof, in the manufactureof a medicament, preferably for lowering the LDL-cholesterol level inblood in a subject in need thereof.

In some aspects of the uses or methods of the invention, saidPCSK9-related disorder is selected from the group consisting of elevatedcholesterol, a condition associated with elevated LDL-cholesterol, e.g.,a lipid disorder (e.g., hyperlipidemia, type I, type II, type III, typeIV, or type V hyperlipidemia, secondary hypertriglyceridemia,hypercholesterolemia, familial hypercholesterolemia, xanthomatosis,cholesterol acetyltransferase deficiency), arteriosclerotic conditions(e.g., atherosclerosis), coronary artery disease, and cardiovasculardisease.

In yet another aspect, the present invention provides the use of anantigenic PCSK9 peptide of the invention or of an immunogeniccomposition or a pharmaceutical composition thereof, in the manufactureof a medicament for treating or alleviating diseases where anup-regulation of the LDL receptor or an inhibition of the interactionbetween PCSK9 and the LDL receptor is beneficial.

In yet another aspect, the present invention provides the use of anantigenic PCSK9 peptide of the invention or of an immunogeniccomposition or a pharmaceutical composition thereof, in the manufactureof a medicament for the treatment of Alzheimer's disease.

In other aspects of the uses or methods of the invention, said subjectis a mammal, preferably a human subject.

In still other aspects of the uses or methods of the invention, saidsubject suffers from said PSCK9-related disorder. Alternatively, saidsubject is at risk of suffering from said PCSK9-related disorder, e.g.,due to the presence of one or more risk factors (e.g., hypertension,cigarette smoking, diabetes, obesity, or hyperhomocysteinemia).

The antigenic PCSK9 peptide of the invention or an immunogeniccomposition or a pharmaceutical composition thereof are useful forsubjects who are intolerant to therapy with another cholesterol-reducingagent, or for whom therapy with another cholesterol-reducing agent hasproduced inadequate results (e.g., subjects who experience insufficientLDL-c reduction on statin therapy). The antigenic PCSK9 peptide of theinvention described herein can be administered to a subject withelevated LDL-cholesterol.

Preferably a subject with elevated cholesterol is a human subject withtotal plasma cholesterol levels of 200 mg/dl or greater. Preferably asubject with elevated cholesterol is a human subject withLDL-cholesterol levels of 160 mg/dl or greater.

Total plasma cholesterol levels and LDL-cholesterol levels are measuredusing standard methods on blood samples obtained after an appropriatefast. Protocols to measure total plasma cholesterol levels andLDL-cholesterol levels are well-known to the man skilled in the art.

In one embodiment the antigenic PCSK9 peptide or an immunogeniccomposition or a pharmaceutical composition thereof is administeredtogether with another agent, the two can be administered sequentially ineither order or simultaneously. In some embodiments, an antigenic PCSK9peptide or an immunogenic composition or a pharmaceutical compositionthereof is administered to a subject who is also receiving therapy witha second agent (e.g., a second cholesterol-reducing agent). Cholesterolreducing agents include statins, bile acid sequestrants, niacin, fibricacid derivatives, and long chain alpha, omego-dicarboxylic acids.Statins inhibit cholesterol synthesis by blocking HMGCoA, a key enzymein cholesterol biosynthesis. Examples of statins are lovastatin,pravastatin, atorvastatin, cerivastatin, fluvastatin, and simvastatin.Bile acid sequestrants interrupt the recycling of bile acids from theintestine to the liver. Examples of these agents are cholestyramine andcolestipol hydrochloride. Examples of fibric acid derivatives areclofibrate and gemfibrozil. Long chain alpha, omego-dicarboxylic acidsare described, e.g., by Bisgaier et al., 1998, J. Lipid Res. 39:17-30;WO 98/30530; U.S. Pat. No. 4,689,344; WO 99/001 16; U.S. Pat. No.5,756,344; U.S. Pat. No. 3,773,946; U.S. Pat. No. 4,689,344; U.S. Pat.No. 4,689,344; U.S. Pat. No. 4,689,344; and U.S. Pat. No. 3,930,024);ethers (see, e.g., U.S. Pat. No. 4,711,896; U.S. Pat. No. 5,756,544;U.S. Pat. No. 6,506,799). Phosphates of dolichol (U.S. Pat. No.4,613,593), and azolidinedione derivatives (U.S. Pat. No. 4,287,200) canalso be used to reduce cholesterol levels. A combination therapy regimenmay be additive, or it may produce synergistic results (e.g., reductionsin cholesterol greater than expected for the combined use of the twoagents). In some embodiments, combination therapy with an antigenicPCSK9 peptide or an immunogenic composition or a pharmaceuticalcomposition thereof and a statin produces synergistic results (e.g.,synergistic reductions in cholesterol). In some subjects, this can allowreduction in statin dosage to achieve the desired cholesterol levels.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Selection of Antigenic PCSK9 Peptides at the PCSK9-EGF-ADomain of the LDL Receptor Interface

The structure of human PCSK9 binding to the EGF-A domain of the LDLreceptor has been solved and published (Kwon et al, PNAS 105, 1820-1825,2008). This structural information (PDB: 3BPS) was used together withinformation from the structure of free PCSK9, PDB: 2P4E (Cunningham etal, Nature Structural & Molecular Biology, 14, 413-419, 2007) to designthe following peptides which would correspond to areas of importance forthe PCSK9-LDL receptor interaction (see FIG. 1).

Peptide 1. ASSDCSTCFV (SEQ ID No: 19) Peptide 2. GTRFHRQASK(SEQ ID No: 63) Peptide 3. IQSDHREIEGRV (SEQ ID No: 109) Peptide 4.SGRDAGVAKGA (SEQ ID No: 153) Peptide 5. SIPWNLERITP (SEQ ID No: 184)

Since peptides 1-4 represent loops in the PCSK9 structure, therespective sequences (SEQ ID Nos 19, 63, 109 and 153) were made withadded Cys, Cys-Gly or Lys linkers to allow coupling via both ends to theVLP carrier to provide a conformational mimetic of the natural loopstructure (VR_9.1 to VR_9.4 in Table 1). In addition, cyclised versionsof peptides 2-4 were also made (VR_9.6 to VR_9.9 in Table 1) whichprovided a Cys residue for coupling to VLPs. Peptide 1 was made with aLys-Gly-Gly N-terminal linker for coupling purposes so that the two Cysresidues were free to disulphide bond as they do in the native PCSK9structure. Peptide 5 represents the N-terminal of the mature processedform of human PCSK9 and was coupled via a C-terminally added cysteineresidue to allow the N-terminus to be free for antibody recognition(VR_9.5 in Table 1). The following table (Table 1) describes 9 peptidesgenerated for evaluation as vaccine candidates.

TABLE 1  Peptide Sequences Peptide Sequence SEQ ID No VR_9.1 KGGASSDCSTCFV 313 VR_9.2 CG GTRFHRQASK C 314 VR_9.3 CG IQSDHREIEGRV C 315VR_9.4 C SGRDAGVAKGA C 316 VR_9.5 SIPWNLERITP C 317 VR_9.6 ASK- Cys(H)-GDGTRFHRQ 318 VR_9.7 A G-Cys-(H) -GTRFHRQ 319 VR_9.8 GRV -Cys(H)-IQSDHREIE 320 VR_9.9 AGVAKGA G-Cvs(H) -SGRD 321 Underscore indicatescysteine residues assed for conjugation purposes and double underscoreindicates a GC or KGG linker.

Example 2 Preparation of Peptide-VLP Conjugates for Evaluation asVaccine Candidates

The peptides were synthesised using a standard Fmoc protocol on CLEARamide resin. The amino acid coupling reactions were carried out using 5fold excess of Fmoc-protected amino acid activated with 1 eq of HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) in the presence of HOBt (hydroxybenzotriazole) andNMM (N-methylmorpholine). The deprotection of Fmoc group was achievedwith 20% piperidine/DMF. Resin-bound peptide was then cleaved and sidechain protecting groups removed simultaneously with Reagent D(TFA/H2O/DODT: 89/3/8). The peptide was made with a free N-terminus andamidated C-terminus. The crude peptide was purified to homogeneity byHPLC using a BEH 130 C18 column and a water/acetonitrile gradient in thepresence of 0.1% TFA. The purified peptide was vacuum-dried using alyophilizer. The peptide was analyzed using mass-spectrometry (LC-MS)and gave satisfactory data.

The Qβ VLP used in this study was produced by bacterial E. Colifermentation in a BL21 (DE3) strain incorporating a pET28 plasmidencoding the 14 kD monomer protein:MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO:638) (Genbank ID: M99039). The fermentationis induced at an OD600 of 0.8 with IPTG and allowed to proceed overnightin terrific broth (TB) with kanamycin. The VLP, which self-assembles inthe host cell, was then purified from the fermentation cell pellet usingthe method described in the patent application EP1736538 with thefollowing differences: after cell disruption, the clarified homogenatewas treated with ammonium sulphate at 50% saturation and the cell pelletrecovered by centrifugation. Then, the pellet was redissolved in HEPESbuffer and dialysed against HEPES buffer before proceeding to the firstcolumn step in the published method. After the ion-exchange column andhydroxylapatite column steps, the material was purified using a furtheranion-exchange column step and sterile filtered to make the final VLPbulk material, which was analysed by size-exclusion chromatography,SDS-PAGE and electron microscopy with acceptable results.

Conjugation of Peptides Through Cysteine Residues:

The Qβ VLP was activated using eitherN-gamma-maleimido-butyryloxy-succinimide ester (GMBS) or the longerSuccinimidyl-6-[β-maleimidopropionamido]hexanoate linking reagent. Theprocedure for the usage of both these reagents was similar: Solidreagent was dissolved in dimethyl sulphoxide (DMSO) and added to the VLPsolution at ≧10-fold molar excess. The activation reaction was allowedto proceed for ≧90 minutes and the solution was then desalted using aNAP-25 desalting column into Dulbeccos Phosphate Buffered Saline (DPBS)with 5 mM EDTA or Dulbeccos Phosphate Buffered Saline (DPBS) that hadbeen modified by the addition of solid NaCl (14.6 g/L). If necessary,the protein solution was concentrated slightly using 10 kD spinmicroconcentrators prior to the next conjugation reaction.

Prior to the conjugation reaction, the peptides were dissolved in analiquot of pH 7.4 DPBS, with 5 mM EDTA as an additive. The concentrationof the peptide in solution was 10 mg/ml. The solubilised peptide wasadded to an aliquot of TCEP immobilised reducing agent (Pierce Chemical)which had been washed in DPBS containing 5 mM EDTA. The aliquot ofpeptides was incubated with mixing in the presence of the TCEP gel forapproximately 1 hour, after which time the aliquot was spun down in amicrofuge and the solid pellet discarded. The reduced peptide-containingsupernatant was added directly to the activated VLP which had beenprepared earlier. One alternative to this procedure however is theaddition of solid peptide directly to the sample of activated Qβ VLP.Both methods work equally well for the generation of peptide-VLPconjugates.

The reaction between the VLPs and the reduced peptides was allowed toproceed for at least thirty minutes with very gentle mixing. At the endof the reaction time each sample was desalted into Dulbeccos PBS (DPBS)using NAP-10 or NAP-25 desalting columns (GE Healthcare). The desaltedconjugated peptides were analysed for protein content using the Bradford(Coomassie Brilliant Blue, Pierce Chemical Co) assay or BCA proteinassay (bicinchoninic acid) (Pierce Chemical Co) as well as by SDS-PAGEand size-exclusion chromatography. The conjugate products were sterilefiltered using a 0.22 μm filter and stored at 2-8° C. until use. Carefulattention was paid to these samples during storage to prevent freezingor exposure to extremes in temperature.

The conjugation of the PCSK9 peptide to CRM197 was performed by usingBAANS (Bromoacetic acid N-hydroxysuccinimide ester, Sigma B8271). CRM197was first activated by reacting with BAANS in 0.1 M sodium carbonate pH8.3 with a molar ratio of 100 in a cold room for 90 minutes. Thereaction mixture was passed through a Zeba desalting column, and theflow-through was collected. The PCSK9 peptide at 10 mg/ml was incubatedwith an equal volume of immobilized TCEP reducing gel (ThermoScientific) at room temperature for 1 hour, and collected aftercentrifugation through a 0.2 μm filter. The activated CRM197 was thenmixed with the treated peptide in the cold room overnight, followed byan extensive dialysis in PBS buffer. The conjugate was recovered,concentrated, and sterilized through a 0.22 μm filter. The proteinconcentration was determined using Coomassie blue assay (ThermoScientific).

Conjugation of Peptides Via Amines

For the conjugation of peptides to Qβ via amine residues, specificallypeptide VR_9.1, the following procedure was followed. Qβ was initiallyderivatised by the addition of solid succinic anhydride at a ≧10-foldmolar excess relative to the VLP monomer. The succinic anhydride wasallowed to dissolve and the derivatisation reaction was allowed proceedfor at least 30 mins. After this time, the sample was then desaltedusing a NAP-25 desalting column into Dulbeccos Phosphate Buffered Saline(DPBS) with 5 mM EDTA. Then, the following reagents were added in theorder listed at a ≧10-fold molar excess relative to the VLP monomer:Solid peptide, N-hydroxysulfosuccinimide and finally1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. Following the addition ofreagents in the order listed above, the sample was incubated at roomtemperature and the reaction was allowed proceed for at least 30 mins,after which time the VLP-peptide conjugate was desalted using NAP-25desalting columns into Dulbeccos Phosphate Buffered Saline (DPBS).

The extent of the conjugation for the VLP-peptide samples was measuredusing SDS-PAGE, and a molecular weight increase was observed for allsamples which is consistent with the addition of the peptide to the VLPprotein monomer. In addition, samples were tested in the HPLCsize-exclusion chromatography assay (using a Tosoh PWXL5000 HPLC column)and found to contain assembled VLP when compared to unconjugated samplesof VLP. VLP-peptide conjugation features are summarized in Table 2.

TABLE 2 VLP-Peptide Conjugates Approximate Amount Percentagesubstitution (ug Activated of peptide yield of peptide per mg of PeptideVLP added added input VLP VLP monomer) VR_9.1 4 mg 5 mg 40-60% 25 ugVR_9.2 3 mg 5 mg 40-60% 125 ug VR_9.3 3 mg 5 mg 40-60% 125 ug VR_9.4 3mg 5 mg 40-60% 125 ug VR_9.5 3 mg 5 mg 40-60% 50 ug VR_9.6 3 mg 5 mg40-60% 125 ug VR_9.7 3 mg 5 mg 40-60% 125 ug VR_9.8 3 mg 5 mg 40-60% 125ug VR_9.9 3 mg 5 mg 40-60% 125 ug VR_9.10 3 mg 5 mg 40-60% 125 ugVR_9.11 3 mg 2 mg 40-60% 125 ug VR_9.12 3 mg 2 mg 40-60% 125 ug VR_9.133 mg 2 mg 40-60% 125 ug VR_9.14 3 mg 2 mg 40-60% 125 ug VR_9.15 3 mg 2mg 40-60% 125 ug VR_9.16 3 mg 2 mg 40-60% 125 ug VR_9.17 10 mg 9.5 mg95% 82 ug VR_9.18 10 mg 8.7 mg 90% 74 ug VR_9.19 10 mg 7.6 mg 80% 58 ugVR_9.20 10 mg 8.7 mg 95% 86 ug VR_9.21 10 mg 8.8 mg 95% 92 ug VR_9.22 10mg 6.6 mg 85% 62 ug VR_9.23 10 mg 10.0 mg 90% 85 ug VR_9.24 10 mg 10.5mg 75% 64 ug VR_9.25 10 mg 5.1 mg 40% 1 ug VR_9.26 10 mg 10.0 mg 60% 123ug VR_9.27 10 mg 9.6 mg 60% 136 ug VR_9.28 10 mg 9.4 mg 65% 153 ugVR_9.29 10 mg 4.2 mg 75% 19 ug VR_9.30 10 mg 4.4 mg 63% 15 ug VR_9.31 10mg 4.4 mg 70% 13 ug VR_9.32 10 mg 4.4 mg 63% 15 ug VR_9.33 10 mg 7.4 mg40% 18 ug VR_9.34 10 mg 7.3 mg 50% 23 ug VR_9.35 7.5 mg 4.3 mg 61% 16 ug*As determined by SDS-PAGE and densitometry calculations

Example 3 PCSK9 Peptide Immunogenicity

This study aimed to evaluate how efficacious peptides conjugated to aQbeta VLP (as detailed in Example 2 above) were in inducing an antibodyresponse that can bind to human and mouse PCSK9. Female Balb/c (6-8weeks) were injected by the intramuscular route (50 microliter volumeinjected into each Tibialis anterior muscle) on days 0, 21 and 42 withVLP-peptide conjugates formulated in Alum with CpG of formula 5′TCGTCGTTTTTCGGTGCTTTT 3′ (SEQ ID NO:589). One group of control mice wasimmunized with VLP coupled to a control (non-PCSK9) peptide followingthe same protocol and a second control group was left unimmunised.Necropsy took place on day 49. At necropsy 400-600 microliter blood wassampled from euthanised mice by cardiac puncture using ananti-coagulant. Blood was centrifuged to separate the plasma, which wasstored frozen until testing.

IgG antibody responses to full length human recombinant PCSK9 proteinwere measured using a colorimetric ELISA method. Serial dilutions wereprepared from sera samples and tested in the assay.

Human PCSK9 ELISA Method 1:

384-well high bind assay plates (Corning International Cat #3700) werecoated with 25 μL/well of human PCSK9 protein stock diluted to 1 μg/mLwith 0.01 M PBS pH 7.4 and incubated on a shaker at RT for 3 hours.After washing ×2 with PBS pH 7.4, plates were blocked using 80 μL/wellof 0.01M PBS/1% BSA, incubated at RT for 1 hour before a final wash ×3with 0.01M PBS pH 7.4/0.05% Tween 20. The following day, an 8 point ½log serial dilution of each sample was prepared starting at 1:25dilution (PBS/1% BSA diluent), 25 μL/well of the serial dilutiontransferred in duplicate into the human PCSK9 coated plate thenincubated shaking at RT for 1.5 hours. After washing ×3 with 0.01 M PBSpH 7.4/0.05% Tween 20, 25 μL/well of Total IgG detection antibody(Rabbit anti-mu IgG-Fc, Cat# A90-130A Bethyl Laboratories) at a 1:6000dilution with 0.01M PBS pH 7.4/1% BSA was added, then incubated shakingat RT for 1 hour. After washing ×5 with 0.01M PBS pH 7.4/0.05% Tween 20,added 25 μL/well Bio-Rad kit goat anti-rabbit horseradish peroxidaseconjugate (Bio-Rad Cat #172-1019) 1:3000 with 0.01M PBS pH 7.4/0.05%Tween 20 pH 7.4, then incubated shaking at RT for 1 hour. After washing×4 with 0.01M PBS pH 7.4/0.05% Tween 20 and then ×1 with 0.01M PBS pH7.4 only, 25 μL/well Mouse Typer HRP Substrate (Bio-Rad Cat #172-1064)were added and the plates were incubated at RT for 30 mins. 25 μL/well2% oxalic acid were added and the absorbance then read at Abs 405 nm.

Mouse PCSK9 ELISA Method 1:

Thermo Immunolon 4HBX 96-well ELISA plates were coated with 100 μl of 1μg/ml recombinant mouse PCSK9 in PBS overnight at 4° C. After washing,plates were blocked with 300 ml PBS/0.5% BSA (Sigma A7030) for 1 hr,washed 4× with 300 μl PBS/0.01% Tween-20 and 100 μl of serial dilutionsof plasma samples (in PBS/0.5% BSA) added. After incubation with gentleshaking for 1 hour at room temperature, plates were washed 4× with 300μl PBS/0.01% Tween-20 and 100 μl of a 1:5000 dilution of goat anti-mouseIgG-HRP (horse radish peroxidase; Pierce 31430) added to each well. Theplates were incubated at room temperature with gentle shaking for 45minutes and then washed ×7 with 300 μl PBS/0.01% Tween-20. 100 μl TMBsubstrate (Sigma T-4444) was then added, the colorimetric reactionstopped after 4 minutes by addition of 2N sulphuric acid and theabsorbance read at 450 nm.

Data Analysis:

Titration curves were plotted for each test sample (sample dilution vsabsorbance). The sample titer (subsequently transformed into reciprocaltiter) was then taken as the serum dilution achieving a cut-off opticaldensity (0.D.) values of 1.0 or 0.5.

Measurement of Plasma/Serum Cholesterol Level

Cholesterol levels in plasma and serum samples were measured using aWAKO Cholesterol E Assay kit (Cat #439-17501) following themanufacturers' instructions. Dilutions of cholesterol standard or testplasma/serum samples (4 μl volume) were added to wells of a 96-wellplate and 196 μl of prepared cholesterol reagent added. The plate wasincubated for 5 minutes at 37° C. and the absorbance of the developedcolour read at 600 nm within 30 minutes.

Measurement of Interaction Between PCSK9 and the Extracellular Domain ofthe LDL Receptor

Interruption of LDLR and PCSK9 binding by mouse plasma was determinedwith TR-FRET assay (time-resolved fluorescence resonance energy transferassay) using fluorophor-labeled LDLR extracellular domain (LDLR-ECD) andfull length wild type PCSK9 protein. LDLR-ECD (R&D system, cat#2148-LD/CF)) was labeled with europium (GE healthcare, Cat#PA99148)according to manufacturer's instruction (at a Eu:LDLR molar ratio 6:1).PCSK9 was biotinylated with Biotin-XX-SSE (Pierce, cat #21237) at aBiotin:PCSK9 molar ratio of 8. The TR-FRET assay was conducted with 5 nMLDLR-Eu+3, 30 nM PCSK9-biotin and 50 nM Alexa Fluor 647 conjugatedstreptavidin (SA647, Invitrogen, cat# S21374) in 20 ul of assay buffer(20 mM Hepes, pH7.0, 150 mM NaCl, 0.1 mM CaCl2 and 0.05% (w/v) BSA) in384-well black plates (Corning, Cat #3676). Serial dilution of mouseplasma were pre-incubated with PCSK9-biotin at RT for 30 minutes inhumidified chamber, followed by mixing with LDLR and streptavidin-SA647.After an additional 60 minute incubation at RT in a humidifiedatmosphere in dark, the plates were read on a Perkin Elmer Victor2 platereader using a 50 μs delay time and a 400 ρs window. Data are reportedas the ratio of the signals at (665 nm/615 nM)×1000.

Results:

as shown in FIG. 2, all peptides used as immunogens were able to induceantibody responses to the intact full-length human PCSK9 protein, someinducing higher responses than others. In all cases these responsescross-reacted with mouse PCSK9 as shown in FIG. 3. FIG. 4 shows thatpeptide VR_9.5 immunization also led to a decrease in plasma cholesterollevels and FIGS. 5 and 6 show that VR_9.5 and VR_9.6 induced serumantibody responses that could inhibit the interaction between PCSK9 andLDL receptors using a fluorescence resonance energy transfer (FRET)assay.

Example 4 Design of Peptides Corresponding to Regions Distinct fromReceptor EGF-A Domain Present in PDB: 3BPS

The LDL receptor is a multidomain protein the extracellular domains ofwhich consist of an N-terminal ligand binding domain, three EGF likerepeats (of which EGF-A is one) a β-propeller domain and an “O linkedsugar domain” (Kwon et al, PNAS 105, 1820-1825, 2008). The PDB file 3GCXdetails the structure of PCSK9 in complex with a soluble form of onlythe EGF-A domain of the LDL receptor, therefore we postulated thatfurther non-obvious interactions may exist between PCSK9 and the LDLreceptor that cannot be deduced from direct analysis of the moleculesrepresented in PDB: 3GCX. Examples of these regions are detailed in FIG.7 and specifically two sequences in PCSK9 were identified that could actas additional putative receptor interfaces (NAQDQPVTLGTL (SEQ ID NO:227)and INEAWFPEDQRVL (SEQ ID NO:263)—see FIG. 8).

SIPWNLERIIP (mouse) (SEQ ID No 332) NAQDQPVTLGTL (SEQ ID No: 227)INEAWFPEDQRVL (SEQ ID No: 263) INMAWFPEDQQVL (mouse) (SEQ ID No 360)

By using murine PCSK9 sequences as found in public databases the murinehomologues were also identified (as displayed in table 3). We alsopostulated that part of the amino acid sequence contained within peptideVR_9.5 (described in Example 1) may also interact with parts of the LDLreceptor not visible in the PDB: 3BPS (FIG. 8). This concept was probedby refining the sequence VR_9.5 by altering the amino acid linker andthe orientation of conjugation. We also identified a peptidecorresponding to the mouse homologue of VR_9.5 (peptide VR_9.10). Theresultant series of peptide sequences from the approach described abovewere modified by the addition of amino acids to permit chemicalconjugation and were evaluated as vaccine candidates (peptides detailedin table 3).

TABLE 3  Peptide sequences Peptide Sequence Species SEQ ID No VR_9.10SIPWNLERIIPC Mouse 322 VR_9.11 CGGSIPWNLERIIP Mouse 323 VR_9.12SIPWNLERIIPGGC Mouse 324 VR_9.13 CGGNAQDQPVTLGTL Mouse & Human 325VR_9.14 NAQDQPVTLGTLGGC Mouse & Human 326 VR_9.15 CGGINMAWFPEDQQVL Mouse327 VR_9.16 INMAWFPEDQQVLGGC Mouse 328 Residues underlined indicateamino acids added for conjugation purposes.

Example 5

Peptides VR_9.10 to VR_9.16 (as well as VR_9.5 for comparison) wereconjugated to Qβ VLP as described in example 2 and used to immunize miceand assess antibody responses to PCSK9 as described in example 3, withan unconjugated VLP used as a control immunogen. As shown in FIGS. 9 and10, all peptides induced antibodies that could recognize intactfull-length mouse PCSK9 in an ELISA assay.

Example 6

On the basis of our observation of cholesterol lowering being induced byimmunization with peptide VR_9.5, representing the N-terminus of themature processed form of human PCSK9 (SEQ ID No: 184), we hypothesizethat, since the cleaved prodomain of immature PCSK9 is known to remainassociated with the mature PCSK9 protein, regions of this prodomain (SEQID No. 329) are also candidate antibody targets for lowing cholesterollevels.

(SEQ ID No: 329) MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDS SVFAQ

Specific, non-restrictive, examples of peptides of interest are foundwithin the C-terminal sequence of the prodomain region and surfaceexposed sequences and loops, including regions containing knownloss-of-function or gain-of-function genetic mutations in humans:

VDYIEEDSSVFAQ (SEQ ID No: 308) RCAKDPWRLPGT (SEQ ID No: 309)AQAARRGYLTKIL (SEQ ID No: 310) GDYEELVLALRSEEDG (SEQ ID No: 311)FLVKMSGDLLELALKLP (SEQ ID No: 312)

The above peptides and truncations thereof are synthesized with N- orC-terminally added linkers (e.g. CGG or GGC), or as cyclised orotherwise conformationally constrained molecules and coupled to Qβ VLPsas described in example 2 and used to immunize mice and assess antibodyresponses to PCSK9 as described in example 3.

Example 7

Peptides VR_9.5 and 9.10 were conjugated to Qβ VLP or to CRM197 asdescribed in example 2 and used to immunize mice (BALB/c or C57BL/6)using TiterMax Gold, alum or alum-CpG as adjuvant. Hepatitis B virusderived peptide (amino acids 28-39 (IPQSLDSWWTSL)) was conjugated to QβVLP or to CRM197 and used as a control immunogen.

The ELISA method used was slightly different from the one disclosed inexample 3: the ELISA method was performed as follows for Human and MousePCSK9 ELISA (Method 2): 384-well high bind assay plates (Greiner bio-one781061) were coated with 25 μL/well of human or mouse PCSK9 proteinstock diluted to 1 μg/mL with 1×PBS pH 7.4 and incubated at 4° C. overnight. The following day, plates were blocked using 25 μL/well of1×PBS/0.05% Tween-20/1% BSA, incubated on a shaker at RT for 1 hour. A10 point ½ log serial dilution of each sample was prepared starting at1:50 or 1:500 dilution (1×PBS/0.05% Tween-20 diluent), 25 μL/well of theserial dilution transferred in duplicate into the human or mouse PCSK9coated plate then incubated shaking at RT for 1 hour. After washing ×3with 1×PBS pH 7.4/0.05% Tween-20, 25 μL/well of Total IgG detectionantibody (Goat Anti-Mouse IgG (γ), HRP, Invitrogen M30107) at a 1:3000dilution with 1×PBS pH 7.4/0.05% Tween-20 was added then incubatedshaking at RT for 1 hour. After washing ×5 with 1×PBS pH 7.4/0.05% Tween20, 25 μL/well Bio-Rad TMB Peroxidase EIA Substrate Kit (Bio-Rad Cat#172-1067) were added and the plates incubated for 15 minutes. 12.5μL/well of 1N sulfuric acid were added and the absorbance then read atAbs 450 nm.

Results:

Peptides VR_9.5 and 9.10 conjugated to Qβ VLP or CRM197 were able toinduce antibody responses to the intact full-length human and mousePCSK9 protein (see FIGS. 11 and 12). FIGS. 13 and 14 and Table 4 alsoshow that peptide VR_9.5 and 9.10 conjugated to different carriers andin the presence of different adjuvants led to a decrease in serumcholesterol levels.

TABLE 4 Total Cholesterol Levels in Immunized Mmice Cholesterol Mouse(mg/dL) Strain Immunogen Carrier Adjuvant(s) N Mean SEM Stats^((a))Stats^((b)) BALB/c Untreated — — 8 106.5 3.0 — — Control peptide VLPAlum + CpG 8 99.0 2.8 P > 0.05 — VR_9.5 VLP Alum + CpG 8 77.5 3.6 P <0.001 P < 0.001 VR_9.5 VLP Alum 8 65.9 2.3 P < 0.001 P < 0.001 VR_9.10VLP Alum + CpG 8 85.3 2.4 P < 0.001 P < 0.05 Control peptide CRM197TiterMax 8 96.6 2.3 P > 0.05 — VR_9.5 CRM197 TiterMax 8 75.0 4.1 P <0.001 P < 0.001 VR_9.10 CRM197 TiterMax 7 70.9 2.8 P < 0.001 P < 0.001C57BL/6 Untreated — — 8 83.4 5.8 — — Control peptide VLP Alum + CpG 881.0 3.7 P > 0.05 — VR_9.5 VLP Alum + CpG 8 59.7 2.8 P < 0.001 P < 0.01VR_9.5 VLP Alum 8 59.5 1.5 P < 0.001 P < 0.01 VR_9.10 VLP Alum + CpG 864.5 2.7 P < 0.01 P < 0.05 Control peptide CRM197 TiterMax 8 71.5 1.7P > 0.05 — VR_9.5 CRM197 TiterMax 8 58.7 4.1 P < 0.001 P > 0.05 VR_9.10CRM197 TiterMax 8 63.8 3.4 P < 0.01 P > 0.05 Stats^((a)): 1-way ANOVAstatistical comparison with Tukey post-hoc test displaying p values oftest groups vs. untreated. Stats^((b)): 1-way ANOVA statisticalcomparison with Tukey post-hoc test displaying p values of test groupsvs. matched control peptide-carrier.

Example 8

Additional peptides (SEQ ID No 312, 420, 421, 422, 423, 425, 426, 427,428, 445, 482, 525 and 563) were chosen from both the prodomain and theC-terminal region of the catalytic domain of PCSK9 for surface exposureand association with gain of function or loss of function mutationsidentified in humans. The resultant series of peptide sequences from theapproach described above were modified by the addition of amino acids topermit chemical conjugation and were evaluated as vaccine candidates(Table 5 peptides 9.23-9.35).

TABLE 5  Peptide sequences SEQ Peptide Sequence Species ID No VR_9.5SIPWNLERITPC Human 317 VR_9.10 SIPWNLERIIPC Mouse 322 VR_9.17SIPWNLERIGGC Human & Mouse 401 VR_9.18 SIPWNLERGGC Human & Mouse 402VR_9.19 SIPWNLEGGC Human & Mouse 403 VR_9.20 CGGSGRDAGVAKGA Human 404VR_9.21 CGGSGRDAGVAKGT Mouse 405 VR_9.22 RDAGVAKGGC Human 406 VR_9.23CSRHLAQASQELQ Human 407 VR_9.24 CRSRPSAKASWVQ Mouse 408 VR_9.25CGGDYEELVLALR Human 409 VR_9.26 CGGDYEELMLALP Mouse 410 VR_9.27LVLALRSEEDGGC Human 411 VR_9.28 LMLALPSQEDGGC Mouse 412 VR_9.29AKDPWRLPGGC Human 413 VR_9.30 SKEAWRLPGGC Mouse 414 VR_9.31 CGGAARRGYLTKHuman 415 VR_9.32 CGGAARRGYVIK Mouse 416 VR_9.33 FLVKMSGDLLELALKLPGGCHuman 417 VR_9.34 FLVKMSSDLLGLALKLPGGC Mouse 418 VR_9.35 CGGEEDSSVFAQHuman 419 Residues underlined indicate amino acids added for conjugationpurposes.

Example 9

Peptides VR_9.17 to VR_9.35 (as well as VR_9.5) were conjugated to QβVLP as described in example 2 and used to immunize mice to assessantibody responses to PCSK9 as described in example 3. Hepatitis B virusderived peptide (amino acids 28-39 (IPQSLDSWWTSL (SEQ ID NO:623)) wasconjugated to Qβ VLP and used as a control immunogen. Method No 2 wasused for the ELISA.

Result:

As shown in FIGS. 15 and 16, most of the peptides conjugated to Qβ VLPwere able to induce antibody responses to intact full length PCSK9. Noantibody response was detected for peptides 9.31 and 9.32. In certaincases (peptides 9.23, 9.24, 9.27, 9.28, 9.29, 9.30, 9.33, and 9.34) theantibody responses were species specific. Immunization with peptides9.5, 9.18, and 9.29 also resulted in decreased serum cholesterol, whileimmunization with peptides 9.17, 9.19, 9.30, 9.31, and 9.32 resulted ina trend to reduced cholesterol (Table 6, FIG. 17).

TABLE 6 Total Cholesterol Levels in Immunized Mice Cholesterol Mouse(mg/dL) Strain Immunogen Carrier Adjuvants N Mean SEM Stats^((a))Stats^((b)) BALB/c Untreated — — 8 77.7 5.5 — — Control peptide VLPAlum + CpG 4 78.1 1.2 P > 0.05 — VR_9.5 VLP Alum + CpG 8 65.8 1.1 P <0.05 P > 0.05 VR_9.17 VLP Alum + CpG 8 70.4 2.1 P > 0.05 P > 0.05VR_9.18 VLP Alum + CpG 8 65.9 1.6 P < 0.05 P > 0.05 VR_9.19 VLP Alum +CpG 8 71.7 1.7 P > 0.05 P > 0.05 VR_9.20 VLP Alum + CpG 7 85.2 4.0 P >0.05 P > 0.05 VR_9.21 VLP Alum + CpG 8 77.1 2.0 P > 0.05 P > 0.05VR_9.22 VLP Alum + CpG 8 84.5 2.6 P > 0.05 P > 0.05 VR_9.23 VLP Alum +CpG 8 79.0 1.8 P > 0.05 P > 0.05 VR_9.24 VLP Alum + CpG 8 76.9 2.1 P >0.05 P > 0.05 VR_9.25 VLP Alum + CpG 8 77.3 2.5 P > 0.05 P > 0.05VR_9.26 VLP Alum + CpG 8 79.2 2.2 P > 0.05 P > 0.05 VR_9.27 VLP Alum +CpG 8 71.8 3.4 P > 0.05 P > 0.05 VR_9.28 VLP Alum + CpG 8 76.6 2.2 P >0.05 P > 0.05 VR_9.29 VLP Alum + CpG 8 67.0 1.6 P < 0.05 P > 0.05VR_9.30 VLP Alum + CpG 8 70.4 2.3 P > 0.05 P > 0.05 VR_9.31 VLP Alum +CpG 8 71.0 1.7 P > 0.05 P > 0.05 VR_9.32 VLP Alum + CpG 8 72.8 1.0 P >0.05 P > 0.05 VR_9.33 VLP Alum + CpG 8 75.5 2.2 P > 0.05 P > 0.05VR_9.34 VLP Alum + CpG 8 76.0 2.3 P > 0.05 P > 0.05 VR_9.35 VLP Alum +CpG 8 74.5 2.2 P > 0.05 P > 0.05 Stats^((a)): 1-way ANOVA statisticalcomparison with Bonferroni post-hoc test displaying p values of testgroups vs. untreated group. Stats^((b)): 1-way ANOVA statisticalcomparison with Bonferroni post-hoc test displaying p values of testgroups vs. control peptide group.

Sequence listing: SEQ ID No 1 IGASSDCSTCFVS SEQ ID No 2 IGASSDCSTCFVSEQ ID No 3 IGASSDCSTCF SEQ ID No 4 IGASSDCSTC SEQ ID No 5 IGASSDCSTSEQ ID No 6 IGASSDCS SEQ ID No 7 IGASSDC SEQ ID No 8 IGASSD SEQ ID No 9IGASS SEQ ID No 10  GASSDCSTCFVS SEQ ID No 11  GASSDCSTCFV SEQ ID No 12 GASSDCSTCF SEQ ID No 13  GASSDCSTC SEQ ID No 14  GASSDCST SEQ ID No 15 GASSDCS SEQ ID No 16  GASSDC SEQ ID No 17  GASSD SEQ ID No 18  ASSDCSTCFVS SEQ ID No 19   ASSDCSTCFV SEQ ID No 20   ASSDCSTCFSEQ ID No 21   ASSDCSTC SEQ ID No 22   ASSDCST SEQ ID No 23   ASSDCSSEQ ID No 24   ASSDC SEQ ID No 25    SSDCSTCFVS SEQ ID No 26   SSDCSTCFV SEQ ID No 27    SSDCSTCF SEQ ID No 28    SSDCSTCSEQ ID No 29    SSDCST SEQ ID No 30    SSDCS SEQ ID No 31     SDCSTCFVSSEQ ID No 32     SDCSTCFV SEQ ID No 33     SDCSTCF SEQ ID No 34    SDCSTC SEQ ID No 35     SDCST SEQ ID No 36      DCSTCFVSSEQ ID No 37      DCSTCFV SEQ ID No 38      DCSTCF SEQ ID No 39     DCSTC SEQ ID No 40       CSTCFVS SEQ ID No 41       CSTCFVSEQ ID No 42       CSTCF SEQ ID No 43        STCFVS SEQ ID No 44       STCFV SEQ ID No 45         TCFVS SEQ ID No 46 EDGTRFHRQASKCDSSEQ ID No 47 EDGTRFHRQASKCD SEQ ID No 48 EDGTRFHRQASKC SEQ ID No 49EDGTRFHRQASK SEQ ID No 50 EDGTRFHRQAS SEQ ID No 51 EDGTRFHRQASEQ ID No 52 EDGTRFHRQ SEQ ID No 53  DGTRFHRQASKCDS SEQ ID No 54 DGTRFHRQASKCD SEQ ID No 55  DGTRFHRQASKC SEQ ID No 56  DGTRFHRQASKSEQ ID No 57  DGTRFHRQAS SEQ ID No 58  DGTRFHRQA SEQ ID No 59  DGTRFHRQSEQ ID No 60   GTRFHRQASKCDS SEQ ID No 61   GTRFHRQASKCD SEQ ID No 62  GTRFHRQASKC SEQ ID No 63   GTRFHRQASK SEQ ID No 64   GTRFHRQASSEQ ID No 65   GTRFHRQA SEQ ID No 66   GTRFHRQ SEQ ID No 67   TRFHRQASKCDS SEQ ID No 68    TRFHRQASKCD SEQ ID No 69    TRFHRQASKCSEQ ID No 70    TRFHRQASK SEQ ID No 71    TRFHRQAS SEQ ID No 72   TRFHRQA SEQ ID No 73    TRFHRQ SEQ ID No 74     RFHRQASKCDSSEQ ID No 75     RFHRQASKCD SEQ ID No 76     RFHRQASKC SEQ ID No 77    RFHRQASK SEQ ID No 78     RFHRQAS SEQ ID No 79     RFHRQASEQ ID No 80     RFHRQ SEQ ID No 81      FHRQASKCDS SEQ ID No 82     FHRQASKCD SEQ ID No 83      FHRQASKC SEQ ID No 84      FHRQASKSEQ ID No 85      FHRQAS SEQ ID No 86      FHRQA SEQ ID No 87      HRQASKCDS SEQ ID No 88       HRQASKCD SEQ ID No 89       HRQASKCSEQ ID No 90       HRQASK SEQ ID No 91       HRQAS SEQ ID No 92       RQASKCDS SEQ ID No 93        RQASKCD SEQ ID No 94        RQASKCSEQ ID No 95        RQASK SEQ ID No 96         QASKCDS SEQ ID No 97        QASKCD SEQ ID No 98         QASKC SEQ ID No 99          ASKCDSSEQ ID No 100          ASKCD SEQ ID No 101           SKCDS SEQ ID No 102          SIQSDHREIEGRVM SEQ ID No 103           SIQSDHREIEGRVSEQ ID No 104           SIQSDHREIEGR SEQ ID No 105           SIQSDHREIEGSEQ ID No 106           SIQSDHREIE SEQ ID No 107           SIQSDHREISEQ ID No 108            IQSDHREIEGRVM SEQ ID No 109           IQSDHREIEGRV SEQ ID No 110            IQSDHREIEGRSEQ ID No 111            IQSDHREIEG SEQ ID No 112            IQSDHREIESEQ ID No 113            IQSDHREI SEQ ID No 114             QSDHREIEGRVMSEQ ID No 115             QSDHREIEGRV SEQ ID No 116            QSDHREIEGR SEQ ID No 117             QSDHREIEG SEQ ID No 118            QSDHREIE SEQ ID No 119             QSDHREI SEQ ID No 120             SDHREIEGRVM SEQ ID No 121              SDHREIEGRVSEQ ID No 122              SDHREIEGR SEQ ID No 123              SDHREIEGSEQ ID No 124              SDHREIE SEQ ID No 125              SDHREISEQ ID No 126               DHREIEGRVM SEQ ID No 127              DHREIEGRV SEQ ID No 128               DHREIEGRSEQ ID No 129               DHREIEG SEQ ID No 130               DHREIESEQ ID No 131               DHREI SEQ ID No 132                HREIEGRVMSEQ ID No 133                HREIEGRV SEQ ID No 134               HREIEGR SEQ ID No 135                HREIEG SEQ ID No 136               HREIE SEQ ID No 137                 REIEGRVMSEQ ID No 138                 REIEGRV SEQ ID No 139                REIEGR SEQ ID No 140                 REIEG SEQ ID No 141                 EIEGRVM SEQ ID No 142                  EIEGRVSEQ ID No 143                  EIEGR SEQ ID No 144                  IEGRVM SEQ ID No 145                   IEGRVSEQ ID No 146                    EGRVM SEQ ID No 147 VSGRDAGVAKGASSEQ ID No 148 VSGRDAGVAKGA SEQ ID No 149 VSGRDAGVAKG SEQ ID No 150VSGRDAGVAK SEQ ID No 151 VSGRDAGVA SEQ ID No 152  SGRDAGVAKGASSEQ ID No 153  SGRDAGVAKGA SEQ ID No 154  SGRDAGVAKG SEQ ID No 155 SGRDAGVAK SEQ ID No 156  SGRDAGVA SEQ ID No 157   GRDAGVAKGASSEQ ID No 158   GRDAGVAKGA SEQ ID No 159   GRDAGVAKG SEQ ID No 160  GRDAGVAK SEQ ID No 161   GRDAGVA SEQ ID No 162    RDAGVAKGASSEQ ID No 163    RDAGVAKGA SEQ ID No 164    RDAGVAKG SEQ ID No 165   RDAGVAK SEQ ID No 166    RDAGVA SEQ ID No 167     DAGVAKGASSEQ ID No 168     DAGVAKGA SEQ ID No 169     DAGVAKG SEQ ID No 170    DAGVAK SEQ ID No 171     DAGVA SEQ ID No 172      AGVAKGASSEQ ID No 173      AGVAKGA SEQ ID No 174      AGVAKG SEQ ID No 175     AGVAK SEQ ID No 176       GVAKGAS SEQ ID No 177       GVAKGASEQ ID No 178       GVAKG SEQ ID No 179        VAKGAS SEQ ID No 180       VAKGA SEQ ID No 181         AKGAS SEQ ID No 182 SIPWNLERITPPRSEQ ID No 183 SIPWNLERITPP SEQ ID No 184 SIPWNLERITP SEQ ID No 185SIPWNLERIT SEQ ID No 186 SIPWNLERI SEQ ID No 187 SIPWNLER SEQ ID No 188SIPWNLE SEQ ID No 189 SIPWNL SEQ ID No 190 SIPWN SEQ ID No 191 IPWNLERITPPR SEQ ID No 192  IPWNLERITPP SEQ ID No 193  IPWNLERITPSEQ ID No 194  IPWNLERIT SEQ ID No 195  IPWNLERI SEQ ID No 196  IPWNLERSEQ ID No 197  IPWNLE SEQ ID No 198  IPWNL SEQ ID No 199   PWNLERITPPRSEQ ID No 200   PWNLERITPP SEQ ID No 201   PWNLERITP SEQ ID No 202  PWNLERIT SEQ ID No 203   PWNLERI SEQ ID No 204   PWNLER SEQ ID No 205  PWNLE SEQ ID No 206    WNLERITPPR SEQ ID No 207    WNLERITPPSEQ ID No 208    WNLERITP SEQ ID No 209    WNLERIT SEQ ID No 210   WNLERI SEQ ID No 211    WNLER SEQ ID No 212     NLERITPPRSEQ ID No 213     NLERITPP SEQ ID No 214     NLERITP SEQ ID No 215    NLERIT SEQ ID No 216     NLERI SEQ ID No 217      LERITPPRSEQ ID No 218      LERITPP SEQ ID No 219      LERITP SEQ ID No 220     LERIT SEQ ID No 221       ERITPPR SEQ ID No 222       ERITPPSEQ ID No 223       ERITP SEQ ID No 224        RITPPR SEQ ID No 225       RITPP SEQ ID No 226         ITPPR SEQ ID No 227 NAQDQPVTLGTLSEQ ID No 228 NAQDQPVTLGT SEQ ID No 229 NAQDQPVTLG SEQ ID No 230NAQDQPVTL SEQ ID No 231 NAQDQPVT SEQ ID No 232 NAQDQPV SEQ ID No 233NAQDQP SEQ ID No 234 NAQDQ SEQ ID No 235  AQDQPVTLGTL SEQ ID No 236 AQDQPVTLGT SEQ ID No 237  AQDQPVTLG SEQ ID No 238  AQDQPVTLSEQ ID No 239  AQDQPVT SEQ ID No 240  AQDQPV SEQ ID No 241  AQDQPSEQ ID No 242   QDQPVTLGTL SEQ ID No 243   QDQPVTLGT SEQ ID No 244  QDQPVTLG SEQ ID No 245   QDQPVTL SEQ ID No 246   QDQPVT SEQ ID No 247  QDQPV SEQ ID No 248    DQPVTLGTL SEQ ID No 249    DQPVTLGTSEQ ID No 250    DQPVTLG SEQ ID No 251    DQPVTL SEQ ID No 252    DQPVTSEQ ID No 253     QPVTLGTL SEQ ID No 254     QPVTLGT SEQ ID No 255    QPVTLG SEQ ID No 256     QPVTL SEQ ID No 257      PVTLGTLSEQ ID No 258      PVTLGT SEQ ID No 259      PVTLG SEQ ID No 260      VTLGTL SEQ ID No 261       VTLGT SEQ ID No 262        TLGTLSEQ ID No 263 INEAWFPEDQRVL SEQ ID No 264 INEAWFPEDQRV SEQ ID No 265INEAWFPEDQR SEQ ID No 266 INEAWFPEDQ SEQ ID No 267 INEAWFPEDSEQ ID No 268 INEAWFPE SEQ ID No 269 INEAWFP SEQ ID No 270 INEAWFSEQ ID No 271 INEAW SEQ ID No 272  NEAWFPEDQRVL SEQ ID No 273 NEAWFPEDQRV SEQ ID No 274  NEAWFPEDQR SEQ ID No 275  NEAWFPEDQSEQ ID No 276  NEAWFPED SEQ ID No 277  NEAWFPE SEQ ID No 278  NEAWFPSEQ ID No 279  NEAWF SEQ ID No 280   EAWFPEDQRVL SEQ ID No 281  EAWFPEDQRV SEQ ID No 282   EAWFPEDQR SEQ ID No 283   EAWFPEDQSEQ ID No 284   EAWFPED SEQ ID No 285   EAWFPE SEQ ID No 286   EAWFPSEQ ID No 287    AWFPEDQRVL SEQ ID No 288    AWFPEDQRV SEQ ID No 289   AWFPEDQR SEQ ID No 290    AWFPEDQ SEQ ID No 291    AWFPEDSEQ ID No 292    AWFPE SEQ ID No 293      WFPEDQRVL SEQ ID No 294     WFPEDQRV SEQ ID No 295      WFPEDQR SEQ ID No 296      WFPEDQSEQ ID No 297      WFPED SEQ ID No 298       FPEDQRVL SEQ ID No 299      FPEDQRV SEQ ID No 300       FPEDQR SEQ ID No 301       FPEDQSEQ ID No 302        PEDQRVL SEQ ID No 303        PEDQRV SEQ ID No 304       PEDQR SEQ ID No 305         EDQRVL SEQ ID No 306         EDQRVSEQ ID No 307          DQRVL SEQ ID No 308 VDYIEEDSSVFAQ SEQ ID No 309RCAKDPWRLPGT SEQ ID No 310 AQAARRGYLTKIL SEQ ID No 311 GDYEELVLALRSEEDGSEQ ID No 312 FLVKMSGDLLELALKLP SEQ ID No 313 KGGASSDCSTCFVSEQ ID No 314 CGGTRFHRQASKC SEQ ID No 315 CGIQSDHREIEGRVC SEQ ID No 316CSGRDAGVAKGAC SEQ ID No 317 SIPWNLERITPC SEQ ID No 318 ASKCGDGTRFHRQSEQ ID No 319 AGCGTRFHRQ SEQ ID No 320 GRVCIQSDHREIE SEQ ID No 321AGVAKGAGCSGRD SEQ ID No 322 SIPWNLERIIPC SEQ ID No 323 CGGSIPWNLERIIPSEQ ID No 324 SIPWNLERIIPGGC SEQ ID No 325 CGGNAQDQPVTLGTL SEQ ID No 326NAQDQPVTLGTLGGC SEQ ID No 327 CGGINMAWFPEDQQVL SEQ ID No 328NMAWFPEDQQVLGGC SEQ ID No 329 MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIE EDSSVFAQ SEQ ID No 330SIPWNLERIIPAW SEQ ID No 331 SIPWNLERIIPA SEQ ID No 332 SIPWNLERIIPSEQ ID No 333 SIPWNLERII SEQ ID No 334  IPWNLERIIPAW SEQ ID No 335 IPWNLERIIPAW SEQ ID No 336  IPWNLERIIP SEQ ID No 337  IPWNLERIISEQ ID No 338   PWNLERIIPAW SEQ ID No 339   PWNLERIIPA SEQ ID No 340  PWNLERIIP SEQ ID No 341   PWNLERII SEQ ID No 342    WNLERIIPAWSEQ ID No 343    WNLERIIPA SEQ ID No 344    WNLERIIP SEQ ID No 345   WNLERII SEQ ID No 346     NLERIIPAW SEQ ID No 347     NLERIIPASEQ ID No 348     NLERIIP SEQ ID No 349     NLERII SEQ ID No 350     LERIIPAW SEQ ID No 351      LERIIPA SEQ ID No 352      LERIIPSEQ ID No 353      LERII SEQ ID No 354       ERIIPAW SEQ ID No 355      ERIIPA SEQ ID No 356       ERIIP SEQ ID No 357        RIIPAWSEQ ID No 358        RIIPA SEQ ID No 359         IIPAW SEQ ID No 360INMAWFPEDQQVL SEQ ID No 361 INMAWFPEDQQV SEQ ID No 362 INMAWFPEDQQSEQ ID No 363 INMAWFPEDQ SEQ ID No 364 INMAWFPED SEQ ID No 365 INMAWFPESEQ ID No 366 INMAWFP SEQ ID No 367 INMAWF SEQ ID No 368 INMAWSEQ ID No 369  NMAWFPEDQQVL SEQ ID No 370  NMAWFPEDQQV SEQ ID No 371 NMAWFPEDQQ SEQ ID No 372  NMAWFPEDQ SEQ ID No 373  NMAWFPEDSEQ ID No 374  NMAWFPE SEQ ID No 375  NMAWFP SEQ ID No 376  NMAWFSEQ ID No 377   MAWFPEDQQVL SEQ ID No 378   MAWFPEDQQV SEQ ID No 379  MAWFPEDQQ SEQ ID No 380   MAWFPEDQ SEQ ID No 381   MAWFPEDSEQ ID No 382   MAWFPE SEQ ID No 383   MAWFP SEQ ID No 384    AWFPEDQQVLSEQ ID No 385    AWFPEDQQV SEQ ID No 386    AWFPEDQQ SEQ ID No 387    WFPEDQQVL SEQ ID No 388     WFPEDQQV SEQ ID No 389     WFPEDQQSEQ ID No 390      FPEDQQVL SEQ ID No 391      FPEDQQV SEQ ID No 392     FPEDQQ SEQ ID No 393       PEDQQVL SEQ ID No 394       PEDQQVSEQ ID No 395       PEDQQ SEQ ID No 396        EDQQVL SEQ ID No 397       EDQQV SEQ ID No 398         DQQVL SEQ ID No 399MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHL AQASQELQ SEQ ID No 400MGTHCSAWLRWPLLPLLPPLLLLLLLLCPTGAGAQDEDGDYEELMLALPSQEDGLADEAAHVATATFRRCSKEAWRLPGTYIVVLMEETQRLQIEQTAHRLQTRAARRGYVIKVLHIFYDLFPGFLVKMSSDLLGLALKLPHVEYIEEDSFVFAQSIPWNLERIIPAWHQTEEDRSPDGSSQVEVYLLDTSIQGAHREIEGRVTITDFNSVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGTSLHSLRVLNCQGKGTVSGTLIGLEFIRKSQLIQPSGPLVVLLPLAGGYSRILNAACRHLARTGVVLVAAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGKDIIGASSDCSTCFMSQSGTSQAAAHVAGIVARMLSREPTLTLAELRQRLIHFSTKDVINMAWFPEDQQVLTPNLVATLPPSTHETGGQLLCRTVWSAHSGPTRTATATARCAPEEELLSCSSFSRSGRRRGDWIEAIGGQQVCKALNAFGGEGVYAVARCCLVPRANCSIHNTPAARAGLETHVHCHQKDHVLTGCSFHWEVEDLSVRRQPALRSRRQPGQCVGHQAASVYASCCHAPGLECKIKEHGISGPSEQVTVACEAGWTLTGCNVLPGASLTLGAYSVDNLCVARVHDTARADRTSGEATVAAAICCRS RPSAKASWVQ SEQ ID No 401SIPWNLERIGGC SEQ ID No 402 SIPWNLERGGC SEQ ID No 403 SIPWNLEGGCSEQ ID No 404 CGGSGRDAGVAKGA SEQ ID No 405 CGGSGRDAGVAKGT SEQ ID No 406RDAGVAKGGC SEQ ID No 407 CSRHLAQASQELQ SEQ ID No 408 CRSRPSAKASWVQSEQ ID No 409 CGGDYEELVLALR SEQ ID No 410 CGGDYEELMLALP SEQ ID No 411LVLALRSEEDGGC SEQ ID No 412 LMLALPSQEDGGC SEQ ID No 413 AKDPWRLPGGCSEQ ID No 414 SKEAWRLPGGC SEQ ID No 415 CGGAARRGYLTK SEQ ID No 416CGGAARRGYVIK SEQ ID No 417 FLVKMSGDLLELALKLPGGC SEQ ID No 418FLVKMSSDLLGLALKLPGGC SEQ ID No 419 CGGEEDSSVFAQ SEQ ID No 420SRHLAQASQELQ SEQ ID No 421 DYEELVLALR SEQ ID No 422 LVLALRSEEDGSEQ ID No 423 EEDSSVFAQ SEQ ID No 424 SGRDAGVAKGT SEQ ID No 425RSRPSAKASWVQ SEQ ID No 426 GDYEELMLALP SEQ ID No 427 LMLALPSQEDSEQ ID No 428 FLVKMSSDLLGLALKLP SEQ ID No 429 RCAKDPWRLPG SEQ ID No 430RCAKDPWRLP SEQ ID No 431 RCAKDPWRL SEQ ID No 432 RCAKDPWR SEQ ID No 433RCAKDPW SEQ ID No 434 RCAKDP SEQ ID No 435 RCAKD SEQ ID No 436 CAKDPWRLPGT SEQ ID No 437  CAKDPWRLPG SEQ ID No 438  CAKDPWRLPSEQ ID No 439  CAKDPWRL SEQ ID No 440  CAKDPWR SEQ ID No 441  CAKDPWSEQ ID No 442  CAKDP SEQ ID No 443   AKDPWRLPGT SEQ ID No 444  AKDPWRLPG SEQ ID No 445   AKDPWRLP SEQ ID No 446   AKDPWRLSEQ ID No 447   AKDPWR SEQ ID No 448   AKDPW SEQ ID No 449    KDPWRLPGTSEQ ID No 450    KDPWRLPG SEQ ID No 451    KDPWRLP SEQ ID No 452   KDPWRL SEQ ID No 453    KDPWR SEQ ID No 454     DPWRLPGTSEQ ID No 455     DPWRLPG SEQ ID No 456     DPWRLP SEQ ID No 457    DPWRL SEQ ID No 458      PWRLPGT SEQ ID No 459      PWRLPGSEQ ID No 460      PWRLP SEQ ID No 461       WRLPGT SEQ ID No 462      WRLPG SEQ ID No 464 AQAARRGYLTKI SEQ ID No 465 AQAARRGYLTKSEQ ID No 466 AQAARRGYLT SEQ ID No 467 AQAARRGYL SEQ ID No 468 AQAARRGYSEQ ID No 469 AQAARRG SEQ ID No 470 AQAARR SEQ ID No 471 AQAARSEQ ID No 472  QAARRGYLTKIL SEQ ID No 473  QAARRGYLTKI SEQ ID No 474 QAARRGYLTK SEQ ID No 475  QAARRGYLT SEQ ID No 476  QAARRGYLSEQ ID No 477  QAARRGY SEQ ID No 478  QAARRG SEQ ID No 479  QAARRSEQ ID No 480   AARRGYLTKIL SEQ ID No 481   AARRGYLTKI SEQ ID No 482  AARRGYLTK SEQ ID No 483   AARRGYLT SEQ ID No 484   AARRGYLSEQ ID No 485   AARRGY SEQ ID No 486   AARRG SEQ ID No 487    ARRGYLTKILSEQ ID No 488    ARRGYLTKI SEQ ID No 489    ARRGYLTK SEQ ID No 490   ARRGYLT SEQ ID No 491    ARRGYL SEQ ID No 492    ARRGY SEQ ID No 493    RRGYLTKIL SEQ ID No 494     RRGYLTKI SEQ ID No 495     RRGYLTKSEQ ID No 496     RRGYLT SEQ ID No 497     RRGYL SEQ ID No 498     RGYLTKIL SEQ ID No 499      RGYLTKI SEQ ID No 500      RGYLTKSEQ ID No 501      RGYLT SEQ ID No 502       GYLTKIL SEQ ID No 503      GYLTKI SEQ ID No 504       GYLTK SEQ ID No 505        YLTKILSEQ ID No 506        YLTKI SEQ ID No 507         LTKIL SEQ ID No 508RCSKEAWRLPGT SEQ ID No 509 RCSKEAWRLPG SEQ ID No 510 RCSKEAWRLPSEQ ID No 511 RCSKEAWRL SEQ ID No 512 RCSKEAWR SEQ ID No 513 RCSKEAWSEQ ID No 514 RCSKEA SEQ ID No 515 RCSKE SEQ ID No 516  CSKEAWRLPGTSEQ ID No 517  CSKEAWRLPG SEQ ID No 518  CSKEAWRLP SEQ ID No 519 CSKEAWRL SEQ ID No 520  CSKEAWR SEQ ID No 521  CSKEAW SEQ ID No 522 CSKEA SEQ ID No 523   SKEAWRLPGT SEQ ID No 524   SKEAWRLPGSEQ ID No 525   SKEAWRLP SEQ ID No 526   SKEAWRL SEQ ID No 527   SKEAWRSEQ ID No 528   SKEAW SEQ ID No 529    KEAWRLPGT SEQ ID No 530   KEAWRLPG SEQ ID No 531    KEAWRLP SEQ ID No 532    KEAWRLSEQ ID No 533    KEAWR SEQ ID No 534     EAWRLPGT SEQ ID No 535    EAWRLPG SEQ ID No 536     EAWRLP SEQ ID No 537     EAWRLSEQ ID No 538      AWRLPGT SEQ ID No 539      AWRLPG SEQ ID No 540     AWRLP SEQ ID No 541       WRLPGT SEQ ID No 542       WRLPGSEQ ID No 543        RLPGT SEQ ID No 544 TRAARRGYVIKVL SEQ ID No 545TRAARRGYVIKV SEQ ID No 546 TRAARRGYVIK SEQ ID No 547 TRAARRGYVISEQ ID No 548 TRAARRGYV SEQ ID No 549 TRAARRGY SEQ ID No 550 TRAARRGSEQ ID No 551 TRAARR SEQ ID No 552 TRAAR SEQ ID No 553  RAARRGYVIKVLSEQ ID No 554  RAARRGYVIKV SEQ ID No 555  RAARRGYVIK SEQ ID No 556 RAARRGYVI SEQ ID No 557  RAARRGYV SEQ ID No 558  RAARRGY SEQ ID No 559 RAARRG SEQ ID No 560  RAARR SEQ ID No 561   AARRGYVIKVL SEQ ID No 562  AARRGYVIKV SEQ ID No 563   AARRGYVIK SEQ ID No 564   AARRGYVISEQ ID No 565   AARRGYV SEQ ID No 566   AARRGY SEQ ID No 567   AARRGSEQ ID No 568    ARRGYVIKVL SEQ ID No 569    ARRGYVIKV SEQ ID No 570   ARRGYVIK SEQ ID No 571    ARRGYVI SEQ ID No 572    ARRGYVSEQ ID No 573    ARRGY SEQ ID No 574     RRGYVIKVL SEQ ID No 575    RRGYVIKV SEQ ID No 576     RRGYVIK SEQ ID No 577     RRGYVISEQ ID No 578     RRGYV SEQ ID No 579      RGYVIKVL SEQ ID No 580     RGYVIKV SEQ ID No 581      RGYVIK SEQ ID No 582      RGYVISEQ ID No 583       GYVIKVL SEQ ID No 584       GYVIKV SEQ ID No 585      GYVIK SEQ ID No 586       YVIKVL SEQ ID No 587       YVIKVSEQ ID No 588        VIKVL SEQ ID No 589 TCGTCGTTTTTCGGTGCTTTTSEQ ID No 590 TCGTCGTTTTTCGGTCGTTTT SEQ ID No 591TCGTCGTTTTGTCGTTTTGTCGTT SEQ ID No 592 TCGTCGTTTCGTCGTTTTGTCGTTSEQ ID No 593 TCGTCGTTTTGTCGTTTTTTTCGA SEQ ID No 594TCGCGTCGTTCGGCGCGCGCCG SEQ ID No 595 TCGTCGACGTTCGGCGCGCGCCGSEQ ID No 596 TCGGACGTTCGGCGCGCGCCG SEQ ID No 597 TCGGACGTTCGGCGCGCCGSEQ ID No 598 TCGCGTCGTTCGGCGCGCCG SEQ ID No 599 TCGACGTTCGGCGCGCGCCGSEQ ID No 600 TCGACGTTCGGCGCGCCG SEQ ID No 601 TCGCGTCGTTCGGCGCCGSEQ ID No 602 TCGCGACGTTCGGCGCGCGCCG SEQ ID No 603TCGTCGTTTTCGGCGCGCGCCG SEQ ID No 604 TCGTCGTTTTCGGCGGCCGCCGSEQ ID No 605 TCGTCGTTTTACGGCGCCGTGCCG SEQ ID No 606TCGTCGTTTTCGGCGCGCGCCGT SEQ ID No 607 TCGTCGACGATCGGCGCGCGCCG

The invention claimed is:
 1. A method for alleviating a PCSK9-relateddisorder in an individual, comprising administering to the individual atherapeutically effective amount of an immunogen, wherein the immunogencomprises an antigenic PCSK9 peptide linked to an immunogenic carrier,wherein the antigenic PCSK9 peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NOs:182, 183, 184, 185,186, 187, and 188, wherein the immunogenic carrier is selected from thegroup consisting of CRM197 and a virus-like particle (VLP), and whereinthe PCSK9-related disorder is selected from the group consisting of anateriosclerotic condition, a coronary artery disease, and acardiovascular disease.
 2. The method according to claim 1, wherein theantigenic PCSK9 peptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NOs:182, 183, 184, and
 185. 3. The methodaccording to claim 1, wherein the immunogenic carrier is CRM197.
 4. Themethod according to claim 1, wherein the immunogenic carrier is a VLP.5. The method according to claim 1 wherein the individual is a human. 6.The method according to claim 5, wherein the PCSK9-related disorder is acoronary artery disease.
 7. The method according to claim 5, wherein thePCSK9-related disorder is an ateriosclerotic condition.
 8. The methodaccording to claim 5, wherein the PCSK9-related disorder is acardiovascular disease.
 9. The method according to claim 1, wherein theantigenic PCSK9 peptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NOs:186, 187, and
 188. 10. The methodaccording to claim 9, wherein the antigenic PCSK9 peptide consists ofSEQ ID NO:
 187. 11. The method according to claim 9, wherein theantigenic PCSK9 peptide consists of an amino acid sequence of SEQ ID NO:188.
 12. The method according claim 1, wherein the immunogen furthercomprises an amino acid linker, and wherein: (i) the amino acid linkeris joined to the C-terminus of the antigenic PCSK9 peptide and isselected from the group consisting of GGC, GC, and a cysteine residue(C); and (ii) the antigenic PCSK9 peptide is linked to the immunogeniccarrier through the cysteine residue of the amino acid linker.
 13. Themethod according to claim 12, wherein the amino acid linker is acysteine residue.
 14. The method according to claim 12, wherein theamino acid linker is a GC.
 15. The method according to claim 12, whereinthe amino acid linker is a GGC.
 16. The method according to claim 12,further comprising administering to the individual an adjuvant.
 17. Themethod according to claim 16, wherein the adjuvant is selected fromalum, CpG oligodeoxynucleotide, and QS21.
 18. A method for alleviating aPCSK9-related disorder in an individual, comprising administering to theindividual a therapeutically effective amount of an antigenic PCSK9peptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 317, 401, 402, and 403, wherein thePCSK9-related disorder is selected from the group consisting of anateriosclerotic condition, a coronary artery disease, and acardiovascular disease.
 19. The method according to claim 18, whereinthe antigenic PCSK9 peptide is linked to an immunogenic carrier selectedfrom the group consisting of a VLP and CRM197.
 20. The method accordingto claim 19, wherein the an immunogenic carrier is CRM197.
 21. A methodfor reducing plasma cholesterol levels in an individual, comprisingadministering to the individual a therapeutically effective amount of anantigenic PCSK9 peptide consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 317, 401, 402, and
 403. 22. Themethod according to claim 21, wherein the antigenic PCSK9 peptide islinked to an immunogenic carrier selected from the group consisting of aVLP and CRM197.
 23. The method according to claim 22, wherein theimmunogenic carrier is CRM197.
 24. A method for reducing plasmacholesterol levels in an individual, comprising administering to theindividual a therapeutically effective amount of an immunogen, whereinthe immunogen comprises an antigenic PCSK9 peptide linked to animmunogenic carrier, wherein the antigenic PCSK9 peptide consists of anamino acid sequence selected from the group consisting of SEQ IDNOs:182, 183, 184, 185, 186, 187, and 188, and wherein the immunogeniccarrier is selected form the group consisting of CRM197 and a virus-likeparticle (VLP).
 25. The method according to claim 24, wherein theimmunogenic carrier is CRM197.
 26. The method according claim 24,wherein the immunogen further comprises an amino acid linker, andwherein: (i) the amino acid linker is joined to the C-terminus of theantigenic PCSK9 peptide and is selected from the group consisting ofGGC, GC, and a cysteine residue (C); and (ii) the antigenic PCSK9peptide is linked to the immunogenic carrier through the cysteineresidue of the amino acid linker.
 27. The method according to claim 26,wherein the amino acid linker is a GGC.
 28. The method according toclaim 24, wherein the individual is a human.
 29. The method according toclaim 28, wherein the antigenic PCSK9 peptide consists of an amino acidsequence selected from the group consisting of SEQ ID NOs:182, 183, 184,and
 185. 30. The method according to claim 28, wherein the antigenicPCSK9 peptide consists of an amino acid sequence of SEQ ID NO: 188.